Conventional imaging modalities are unable to depict the early degeneration of articular cartilage in osteoarthritis, especially in small joints. Optical coherence tomography has previously been used successfully in high-resolution imaging of cartilage tissue. This pilot cadaver study demonstrates the use of intra-articular optical coherence tomography in imaging of articular cartilage of the first carpometacarpal joint, producing high resolution images of the articular surface in which cartilage thickness and surface characteristics were assessed. Findings on optical coherence tomography were confirmed with histology. Furthermore, co-registration of optical coherence tomography and computed tomography was used to accurately determine the scanned trajectory and reconstruct a true-scale image overlay.

We used functional photoacoustic microscopy to image diabetes-induced damage to the microvasculature. To produce an animal model for Type 1 diabetes, we used streptozotocin (STZ), which is particularly toxic to the insulin-producing beta cells of the pancreas in mammals. A set number of ND4 Swiss Webster mice received intraperitoneal injections of STZ for five consecutive days at 50 mg/kg. Most mice developed a significant rise in blood glucose level ( ∼ 400 mg/dL) within three weeks of the first injection. Changes in vasculature and hemodynamics were monitored for six weeks. The mouse ear was imaged with an optical-resolution photoacoustic microscope at a main blood vessel branch from the root of the ear. There are noticeable and measurable changes associated with the disease, including decreased vessel diameter and possible occlusion due to vessel damage and polyurea. We also observed an increase in the blood flow speed in the vein and a decrease in the artery, which could be due to compensation for the dehydration and vessel diameter changes. Functional and metabolic parameters such as hemoglobin oxygen saturation, oxygen extraction fraction, and oxygen consumption rate were also measured, but showed no significant change.

Scabies is an intensely pruritic disorder induced by a delayed type hypersensitivity reaction to infestation of the skin by the mite Sarcoptes scabiei. The diagnosis of scabies is established clinically and confirmed by identifying mites or eggs by microscopic examination of scrapings from the skin or by surface microscopy using a dermatoscope. Reflectance-mode confocal microscopy is a novel technique used for noninvasive imaging of skin structures and lesions at a resolution compatible to that of conventional histology. Recently, the technique was employed for the confirmation of the clinical diagnosis of scabies. We demonstrate the first ever documentation of a larva moving freely inside the skin of a patient infected with scabies.

This study is to test anatomic correlates, including connecting cilium (CC) and inner segment (IS) ellipsoid, to the hyper-reflective band visualized by optical coherence tomography (OCT) and commonly attributed to the photoreceptor inner/outer segment (IS/OS) junction. A line-scan OCT (LS-OCT) was constructed to achieve sub-cellular resolution (lateral: ∼ 2  μm; axial: ∼ 4  μm) of excised living frog retinas. An electro-optic phase modulator was employed for rapid and vibration-free phase modulation. Comparison of normalized distance measurements between LS-OCT images and histological images revealed that the dominant source of the signal reported as the IS/OS OCT band actually originates from the IS.

We describe a novel application of spectral-domain phase-sensitive optical coherence tomography (SD PS-OCT) to detect the tiny motions of the middle ear structures, such as the tympanic membrane and ossicular chain, and their morphological features for differential diagnosis of CHL. This technique has the potential to provide meaningful vibration of ossicles with a vibration sensitivity of ∼ 0.5  nm at 1 kHz of acoustic stimulation. To the best of our knowledge, this is the first demonstration of depth-resolved vibration imaging of ossicles with a PS-OCT system at a nanometer scale.

Cardiac myofibers are organized into sheet architectures, which contribute to up to 40% of the heart wall thickening for ejection of blood for circulation. It is important to delineate the sheet architecture for a better understanding of cardiac mechanisms. However, current sheet imaging technologies are limited by fixation-induced dehydration/deformation and low spatial resolution. Here we implemented high-resolution label-free photoacoustic microscopy (PAM) of the myocardial sheet architecture. With high endogenous optical-absorption contrast originating mainly from cytochrome, myoglobin, and melanin, PAM can image the unfixed, unstained and unsliced heart without introducing deformation artifacts. A fresh blood-free mouse heart was imaged by PAM ex vivo. The three-dimensional branching sheets were clearly identified within 150 µm depth. Various morphological parameters were derived from the PAM image. The sheet thickness (80±10  μm) and the cleavage height (11±1  μm) were derived from an undehydrated heart for the first time. Therefore, PAM has the potential for the functional imaging of sheet architecture in ex vivo perfused and viable hearts.

Second-harmonic-generation (SHG) microscopy has emerged as a powerful tool to image unstained living tissues and probe their molecular and supramolecular organization. In this article, we review the physical basis of SHG, highlighting how coherent summation of second-harmonic response leads to the sensitivity of polarized SHG to the three-dimensional distribution of emitters within the focal volume. Based on the physical description of the process, we examine experimental applications for probing the molecular organization within a tissue and its alterations in response to different biomedically relevant conditions. We also describe the approach for obtaining information on molecular conformation based on SHG polarization anisotropy measurements and its application to the study of myosin conformation in different physiological states of muscle. The capability of coupling the advantages of nonlinear microscopy (micrometer-scale resolution in deep tissue) with tools for probing molecular structure in vivo renders SHG microscopy an extremely powerful tool for the advancement of biomedical optics, with particular regard to novel technologies for molecular diagnostic in vivo.

Abstract unavailable.

Obtaining absolute chromophore concentrations from photoacoustic images obtained at multiple wavelengths is a nontrivial aspect of photoacoustic imaging but is essential for accurate functional and molecular imaging. This topic, known as quantitative photoacoustic imaging, is reviewed here. The inverse problems involved are described, their nature (nonlinear and ill-posed) is discussed, proposed solution techniques and their limitations are explained, and the remaining unsolved challenges are introduced.

The use of photoelastic analysis contributes to the rehabilitation of patients with oral-sinus-nasal sequelae, which in turn affect important functions such as chewing, swallowing, and speech. The prosthetic rehabilitation with implant-retained dentures is a suitable treatment option. The purpose of this study was to verify, by using a photoelastic analysis, the stress distribution in implant-retained palatal obturator dentures (relined or not) associated with different attachment systems (O-ring, bar-clip, and bar-clip associated with distally placed O-rings). Two photoelastic models were obtained from an experimental maxillary cast presenting an oral-nasal communication. One model had two 13-mm length implants placed on the left region. A total of eight colorless maxillary obturators were fabricated and subsequently four of them were relined with soft silicone soft, and three had attachment systems associated. The assembly (model/attachment system/prosthesis) was positioned in a circular polariscope and a 100-N load was applied at 10  mm/s. The results showed that the denture relining influenced the distribution and amount of stress on the models. The O-ring group displayed the lowest stress levels, followed by bar-clip system associated with distally placed O-rings and bar-clip groups.

Attenuation effects can be significant in photoacoustic tomography since the generated pressure signals are broadband, and ignoring them may lead to image artifacts and blurring. La Rivière et al. [Opt. Lett. 31(6), pp. 781-783, (2006)] had previously derived a method for modeling the attenuation effect and correcting for it in the image reconstruction. This was done by relating the ideal, unattenuated pressure signals to the attenuated pressure signals via an integral operator. We derive an integral operator relating the attenuated pressure signals to the absorbed optical energy for a planar measurement geometry. The matrix operator relating the two quantities is a function of the temporal frequency, attenuation coefficient and the two-dimensional spatial frequency. We perform singular-value decomposition (SVD) of this integral operator to study the problem further. We find that the smallest singular values correspond to wavelet-like eigenvectors in which most of the energy is concentrated at times corresponding to greater depths in tissue. This allows us to characterize the ill-posedness of recovering the absorbed optical energy distribution at different depths in an attenuating medium. This integral equation can be inverted using standard SVD methods, and the initial pressure distribution can be recovered. We conduct simulations and derive an algorithm for image reconstruction using SVD for a planar measurement geometry. We also study the noise and resolution properties of this image-reconstruction method.

Photoacoustics has recently been proposed as a potential method to guide and/or monitor therapy based on high-intensity focused ultrasound (HIFU). We experimentally demonstrate the creation of a HIFU lesion at the location of an optical absorber, by use of photoacoustic signals emitted by the absorber detected on a dual mode transducer array. To do so, a dedicated ultrasound array intended to both detect photoacoustic waves and emit HIFU with the same elements was used. Such a dual-mode array provides automatically coregistered reference frames for photoacoustic detection and HIFU emission, a highly desired feature for methods involving guidance or monitoring of HIFU by use of photoacoustics. The prototype is first characterized in terms of both photoacoustic and HIFU performances. The probe is then used to perform an idealized scenario of photoacoustic-guided therapy, where photoacoustic signals generated by an absorbing thread embedded in a piece of chicken breast are used to automatically refocus a HIFU beam with a time-reversal mirror and necrose the tissue at the location of the absorber.

Photoacoustic ophthalmoscopy (PAOM) is a newly developed retinal imaging technology that holds promise for both fundamental investigation and clinical diagnosis of several blinding diseases. Hence, integrating PAOM with other existing ophthalmic imaging modalities is important to identify and verify the strengths of PAOM compared with the established technologies and to provide the foundation for more comprehensive multimodal imaging. To this end, we developed a retinal imaging platform integrating PAOM with scanning laser ophthalmoscopy (SLO), spectral-domain optical coherence tomography (SD-OCT), and fluorescein angiography (FA). In the system, all the imaging modalities shared the same optical scanning and delivery mechanisms, which enabled registered retinal imaging from all the modalities. High-resolution PAOM, SD-OCT, SLO, and FA images were acquired in both albino and pigmented rat eyes. The reported in vivo results demonstrate the capability of the integrated system to provide comprehensive anatomic imaging based on multiple optical contrasts.

Because cerebral hypoperfusion brings damage to the brain, prevention of cerebrovascular diseases correlative to hypoperfusion by studying animal models makes great sense. Since complicated cerebrovascular adaptive changes in hypoperfusion could not be revealed only by cerebral blood flow (CBF) velocity imaging, we performed multi-parameter imaging by combining laser speckle imaging and functional photoacoustic microscopy. The changes in CBF, hemoglobin oxygen saturation (SO2), and total hemoglobin concentration (HbT) in single blood vessels of ipsilateral cortex were observed during transient cerebral hypoperfusion by ligating the unilateral common carotid artery in rats. CBF, SO2, and HbT, respectively, decreased to 37±3%, 71±7.5%, and 92±1.3% of baseline in 6 s immediately after occlusion, and then recovered to 77±4.8%, 84±8%, and 96±2% of baseline in 60 s. These parameters presented the decrease with different degree and the following recovery over time after ligation, the recovery of SO2 lagged behind those of CBF and HbT, which had the similar response. The results demonstrated that complete monitoring of both cerebral hemodynamic response and oxygen metabolic changes occurred at the earliest period of cerebral hypoperfusion was possible by using the two image modalities with high temporal and spatial resolution.

We present an integrated photoacoustic and ultrasonic three-dimensional (3-D) volumetric imaging system based on a two-dimensional (2-D) matrix array ultrasound probe. A wavelength-tunable dye laser pumped by a Q-switched Nd:YAG laser serves as the light source and a modified commercial ultrasound imaging system (iU22, Philips Healthcare) with a 2-D array transducer (X7-2, Philips Healthcare) detects both the pulse-echo ultrasound and photoacoustic signals. A multichannel data acquisition system acquires the RF channel data. The imaging system enables rendering of co-registered 3-D ultrasound and photoacoustic images without mechanical scanning. The resolution along the azimuth, elevation, and axial direction are measured to be 0.69, 0.90 and 0.84 mm for photoacoustic imaging. In vivo 3-D photoacoustic mapping of the sentinel lymph node was demonstrated in a rat model using methylene blue dye. These results highlight the clinical potential of 3-D PA imaging for identification of sentinel lymph nodes for cancer staging in humans.

Spectroscopic photoacoustic imaging has the potential to discriminate between normal and lipid-rich atheromatous areas of arterial tissue by exploiting the differences in the absorption spectra of lipids and normal arterial tissue in the 740 to 1400 nm wavelength range. Identification of regions of high lipid concentration would be useful to identify plaques that are likely to rupture (vulnerable plaques). To demonstrate the feasibility of visualizing lipid-rich plaques, samples of human aortas were imaged in forward mode, at wavelengths of 970 and 1210 nm. It was shown that the structure of the arterial wall and the boundaries of lipid-rich plaques obtained from the photoacoustic images were in good agreement with histology. The presence of lipids was also confirmed by comparing the photoacoustic spectra (740 to 1400 nm) obtained in a region within the plaque to the spectral signature of lipids. Furthermore, a lipid-rich plaque was successfully imaged while illuminating the sample through 2.8 mm of blood demonstrating the possibility of implementing the photoacoustic technique in vivo.

The specificity of the hemodynamic response function (HRF) is determined spatially by the vascular architecture and temporally by the evolution of hemodynamic changes. Here, we used functional photoacoustic microscopy (fPAM) to investigate single cerebral blood vessels of rats after left forepaw stimulation. In this system, we analyzed the spatiotemporal evolution of the HRFs of the total hemoglobin concentration (HbT), cerebral blood volume (CBV), and hemoglobin oxygen saturation (SO2). Changes in specific cerebral vessels corresponding to various electrical stimulation intensities and durations were bilaterally imaged with 36×65-μm2 spatial resolution. Stimulation intensities of 1, 2, 6, and 10 mA were applied for periods of 5 or 15 s. Our results show that the relative functional changes in HbT, CBV, and SO2 are highly dependent not only on the intensity of the stimulation, but also on its duration. Additionally, the duration of the stimulation has a strong influence on the spatiotemporal characteristics of the HRF as shorter stimuli elicit responses only in the local vasculature (smaller arterioles), whereas longer stimuli lead to greater vascular supply and drainage. This study suggests that the current fPAM system is reliable for studying relative cerebral hemodynamic changes, as well as for offering new insights into the dynamics of functional cerebral hemodynamic changes in small animals.

We report an investigation of image reconstruction in photoacoustic tomography for objects that possess heterogeneous material and acoustic attenuation distributions. When the object contains materials, such as bone and soft-tissue, that are modeled using power law attenuation models with distinct exponents, we demonstrate that the effects of acoustic attenuation due to the most strongly attenuating material can be compensated for if the attenuation of the other less attenuating material(s) are neglected. Experiments with phantom objects are presented to validated our findings.

Evanescent field sensing methods are currently used to detect many different types of disease markers and biologically important chemicals such as the HER2 breast cancer receptor. Hinoue et al. used Total Internal Reflection Photoacoustic Spectroscopy (TIRPAS) as a method of using the evanescent field to detect an optically opaque dye at a sample interface. Although their methods were successful at detecting dyes, the results at that time did not show a very practical spectroscopic technique, which was due to the less than typical sensitivity of TIRPAS as a spectroscopy modality given the low power ( ∼ 1 to 2 W) lasers being used. Contrarily, we have used an Nd:YAG laser with a five nanosecond pulse that gives peak power of 1 MW coupled with the TIRPAS system to increase the sensitivity of this technique for biological material sensing. All efforts were focused on the eventual detection of the optically absorbing material, hemozoin, which is created as a byproduct of a malarial infection in blood. We used an optically analogous material, β-hematin, to determine the potential for detection in the TIRPAS system. In addition, four properties which control the sensitivity were investigated to increase understanding about the sensor's function as a biosensing method.

Photoacoustic tomography provides the distribution of absorbed optical energy density, which is the product of optical absorption coefficient and optical fluence distribution. We report the experimental investigation of a novel fitting procedure that quantitatively determines the optical absorption coefficient of chromophores. The experimental setup consisted of a hybrid system of a 64-channel photoacoustic imaging system with a frequency-domain diffused optical measurement system. The fitting procedure included a complete photoacoustic forward model and an analytical solution of a target chromophore using the diffusion approximation. The fitting procedure combines the information from the photoacoustic image and the background information from the diffuse optical measurements to minimize the photoacoustic measurements and forward model data and recover the target absorption coefficient quantitatively. 1-cm-cube phantom absorbers of high and low contrasts were imaged at depths of up to 3.0 cm. The fitted absorption coefficient results were at least 80% of their true values. The sensitivities of this fitting procedure to target location, target radius, and background optical properties were also investigated. We found that this fitting procedure was most sensitive to the accurate determination of the target radius and depth. Blood sample in a thin tube of radius 0.58 mm, simulating a blood vessel, was also studied. The photoacoustic images and fitted absorption coefficients are presented. These results demonstrate the clinical potential of this fitting procedure to quantitatively characterize small lesions in breast imaging.

The calibration dependencies of the optoacoustic (OA) transformation efficiency on tissue temperature are obtained for the application in OA temperature monitoring during thermal therapies. Accurate measurement of the OA signal amplitude versus temperature is performed in different ex vivo tissues in the temperature range 25°C to 80°C. The investigated tissues were selected to represent different structural components: chicken breast (skeletal muscle), porcine lard (fatty tissue), and porcine liver (richly perfused tissue). Backward mode of the OA signal detection and a narrow probe laser beam were used in the experiments to avoid the influence of changes in light scattering with tissue coagulation on the OA signal amplitude. Measurements were performed in heating and cooling regimes. Characteristic behavior of the OA signal amplitude temperature dependences in different temperature ranges were described in terms of changes in different structural components of the tissue samples. The accuracy of temperature reconstruction from the obtained calibration dependencies for the investigated tissue types is evaluated.

Using a recently developed reconstruction method for photoacoustic tomography (PAT) valid for a planar measurement geometry parallel to a layered medium, we investigate the effects of shear wave propagation in the solid layer upon the ability to estimate Fourier components of the object. We examine this ability as a function of the thickness of the layer supporting shear waves as well as of the incidence angle of the field in the planewave representation. Examples are used to demonstrate the importance of accounting for shear waves in transcranial PAT. Error measures are introduced to quantify the error found when omitting shear waves from the forward model in PAT.

Absorption coefficient of biological tissue is an important factor for photothermal therapy and photoacoustic imaging. However, its determination remains a challenge. In this paper, we propose a method using focusing photoacoustic imaging technique to quantify the target optical absorption coefficient. It utilizes the ratio of the amplitude of the peak signal from the top boundary of the target to that from the bottom boundary based on wavelet transform. This method is self-calibrating. Factors, such as absolute optical fluence, ultrasound parameters, and Grüneisen parameter, can be canceled by dividing the amplitudes of the two peaks. To demonstrate this method, we quantified the optical absorption coefficient of a target with various concentrations of an absorbing dye. This method is particularly useful to provide accurate absorption coefficient for predicting the outcomes of photothermal interaction for cancer treatment with absorption enhancement.

The detection of ultrasound in photoacoustic tomography (PAT) usually relies on ultrasonic transducers in contact with the biological tissue through a coupling medium. This is a major drawback for important potential applications such as surgery. Here we report the use of a remote optical method, derived from industrial laser-ultrasonics, to detect ultrasound in tissues. This approach enables non-contact PAT (NCPAT) without exceeding laser exposure safety limits. The sensitivity of the method is based on the use of suitably shaped detection laser pulses and a confocal Fabry-Perot interferometer in differential configuration. Reliable image reconstruction is obtained by measuring remotely the surface profile of the tissue with an optical coherence tomography system. The proposed method also allows non-contact ultrasound imaging (US) by applying a second reconstruction algorithm to the data acquired for NCPAT. Endogenous and exogenous inclusions exhibiting optical and acoustic contrasts were detected ex vivo in chicken breast and calf brain specimens. Inclusions down to 0.3 mm in size were detected at depths exceeding 1 cm. The method could expand the scope of photoacoustic and US to in-vivo biomedical applications where contact is impractical.

High-energy and short-duration laser pulses are desirable to improve the photoacoustic image quality when imaging deeply seated lesions. In many clinical applications, the high-energy pulses are coupled to tissue using optical fibers. These pulses can damage fibers if the damage threshold is exceeded. While keeping the total energy under the Food and Drug Administration limit for avoiding tissue damage, it is necessary to reduce the peak intensity and increase the pulse duration for minimizing fiber damage and delivering sufficient light for imaging. We use laser-pulse-stretching to address this problem. An initial 17-ns pulse was stretched to 27 and 37 ns by a ring-cavity laser-pulse-stretching system. The peak power of the 37-ns stretched pulse reduced to 42% of the original, while the fiber damage threshold was increased by 1.5-fold. Three ultrasound transducers centered at 1.3-, 3.5-, and 6-MHz frequencies were simulated, and the results showed that the photoacoustic signal of a 0.5-mm-diameter target obtained with 37-ns pulse was about 98, 91, and 80%, respectively, using the same energy as the 17-ns pulse. Simulations were validated using a broadband hydrophone. Quantitative comparisons of photoacoustic images obtained with three corresponding transducers showed that the image quality was not affected by stretching the pulse.

The induced thermal damage in retinal photocoagulation depends on the temperature increase and the time of irradiation. The temperature rise is unknown due to intraocular variations in light transmission, scattering and grade of absorption in the retinal pigment epithelium (RPE) and the choroid. Thus, in clinical practice, often stronger and deeper coagulations are applied than therapeutically needed, which can lead to extended neuroretinal damage and strong pain perception. This work focuses on an optoacoustic (OA) method to determine the temperature rise in real-time during photocoagulation by repetitively exciting thermoelastic pressure transients with nanosecond probe laser pulses, which are simultaneously applied to the treatment radiation. The temperature-dependent pressure amplitudes are non-invasively detected at the cornea with an ultrasonic transducer embedded in the contact lens. During clinical treatment, temperature courses as predicted by heat diffusion theory are observed in most cases. For laser spot diameters of 100 and 300 μm, and irradiation times of 100 and 200 ms, respectively, peak temperatures range between 70°C and 85°C for mild coagulations. The obtained data look very promising for the realization of a feedback-controlled treatment, which automatically generates preselected and reproducible coagulation strengths, unburdens the ophthalmologist from manual laser dosage, and minimizes adverse effects and pain for the patient.

The ability to noninvasively image embryonic vascular anatomy in mouse models is an important requirement for characterizing the development of the normal cardiovascular system and malformations in the heart and vascular supply. Photoacoustic imaging, which can provide high resolution non invasive images of the vasculature based upon optical absorption by endogenous hemoglobin, is well suited to this application. In this study, photoacoustic images of mouse embryos were obtained ex vivo and in vivo. The images show intricate details of the embryonic vascular system to depths of up to 10 mm, which allowed whole embryos to be imaged in situ. To achieve this, an all-optical photoacoustic scanner and a novel time reversal image reconstruction algorithm, which provide deep tissue imaging capability while maintaining high spatial resolution and contrast were employed. This technology may find application as an imaging tool for preclinical embryo studies in developmental biology as well as more generally in preclinical and clinical medicine for studying pathologies characterized by changes in the vasculature.

Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastastic melanoma cells in blood.

In this study, we develop a novel photoacoustic imaging technique based on gold nanorods (AuNRs) for quantitatively monitoring focused-ultrasound (FUS) induced blood-brain barrier (BBB) opening in a rat model in vivo. This study takes advantage of the strong near-infrared absorption (peak at ∼ 800  nm) of AuNRs and the extravasation tendency from BBB opening foci due to their nano-scale size to passively label the BBB disruption area. Experimental results show that AuNR contrast-enhanced photoacoustic microscopy (PAM) successfully reveals the spatial distribution and temporal response of BBB disruption area in the rat brains. The quantitative measurement of contrast enhancement has potential to estimate the local concentration of AuNRs and even the dosage of therapeutic molecules when AuNRs are further used as nano-carrier for drug delivery or photothermal therapy. The photoacoustic results also provide complementary information to MRI, being helpful to discover more details about FUS induced BBB opening in small animal models.

Laser coagulation is a treatment method for many retinal diseases. Due to variations in fundus pigmentation and light scattering inside the eye globe, different lesion strengths are often achieved. The aim of this work is to realize an automatic feedback algorithm to generate desired lesion strengths by controlling the retinal temperature increase with the irradiation time. Optoacoustics afford non-invasive retinal temperature monitoring during laser treatment. A 75  ns/523  nm Q-switched Nd:YLF laser was used to excite the temperature-dependent pressure amplitudes, which were detected at the cornea by an ultrasonic transducer embedded in a contact lens. A 532 nm continuous wave Nd:YAG laser served for photocoagulation. The ED50 temperatures, for which the probability of ophthalmoscopically visible lesions after one hour in vivo in rabbits was 50%, varied from 63°C for 20 ms to 49°C for 400 ms. Arrhenius parameters were extracted as ΔE = 273  J mol−1 and A = 3·1044  s−1. Control algorithms for mild and strong lesions were developed, which led to average lesion diameters of 162±34  μm and 189±34  μm, respectively. It could be demonstrated that the sizes of the automatically controlled lesions were widely independent of the treatment laser power and the retinal pigmentation.

Contrast-enhanced photoacoustic (PA) imaging has been proposed to identify circulating metastatic cancer cells magnetically trapped in the vasculature. However, its sensitivity is limited by the presence of a strong blood-background signal. This technique can be further improved by the significant suppression of blood background. In the phantom study presented here, significant background suppression is demonstrated with magnetomotive photoacoustic imaging. Magnetic particles with a mean diameter of 10 μm were integrated (concentration of 0.05  mg/ml) into an ink-water solution with an optical absorption coefficient of 5  cm−1 to mimic cells targeted with magnetic nanoparticles and magnetically trapped in the human vasculature. Two mechanically moveable permanent magnets were used to accumulate microparticles in the investigated solution and manipulate them within a thin, 1.6-mm-diameter Teflon tube mimicking a blood vessel. Our results clearly indicate that the undesirable background can be effectively suppressed using the difference of PA images corresponding to different locations of accumulated particles.

We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650  mm3. Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8±103.4  mm2 at the beginning versus 704.8±94.4  mm3 at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9%±4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5±0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

The purpose of this study was to provide the physicochemical interactions at the interfaces between two commercial etch-&-rinse adhesives and human dentin in a simulated moist bond technique. Six dentin specimens were divided into two groups (n = 3) according to the use of two different adhesive systems: (a) 2-hydroxyethylmethacrylate (HEMA) and 4-methacryloxyethyl trimellitate anhydrate (4-META), and (b) HEMA. The Fourier transform infrared photoacoustic spectroscopy was performed before and after dentin treatment with 37% phosphoric acid, with adhesive systems and also for the adhesive systems alone. Acid-conditioning resulted in a decalcification pattern. Adhesive treated spectra subtraction suggested the occurrence of chemical bonding to dentin expressed through modifications of the OH stretching peak (3340  cm−1) and symmetric CH stretching (2900  cm−1) for both adhesives spectra; a decrease of orthophosphate absorption band (1040 to 970  cm−1) for adhesive A and a better resolved complex band formation (1270 to 970  cm−1) for adhesive B were observed. These results suggested the occurrence of chemical bonding between sound human dentin and etch-&-rinse adhesives through a clinical typical condition.

In this study, we demonstrate a method to quantify biomarker expression that uses an exogenous dual-reporter imaging approach to improve tumor signal detection. The uptake of two fluorophores, one nonspecific and one targeted to the epidermal growth factor receptor (EGFR), were imaged at 1 h in three types of xenograft tumors spanning a range of EGFR expression levels (n = 6 in each group). Using this dual-reporter imaging methodology, tumor contrast-to-noise ratio was amplified by >6 times at 1 h postinjection and >2 times at 24 h. Furthermore, by as early as 20 min postinjection, the dual-reporter imaging signal in the tumor correlated significantly with a validated marker of receptor density (P<0.05, r = 0.93). Dual-reporter imaging can improve sensitivity and specificity over conventional fluorescence imaging in applications such as fluorescence-guided surgery and directly approximates the receptor status of the tumor, a measure that could be used to inform choices of biological therapies.

We performed large area nonlinear optical microscopy (NOM) for label-free monitoring of the process of pulmonary melanoma metastasis ex vivo with subcellular resolution in C57BL/6 mice. Multiphoton autofluorescence (MAF) and second harmonic generation (SHG) images of lung tissue are obtained in a volume of ∼ 2.2  mm×2.2  mm×30  μm. Qualitative differences in morphologic features and quantitative measurement of pathological lung tissues at different time points are characterized. We find that combined with morphological features, the quantitative parameters, such as the intensity ratio of MAF and SHG between pathological tissue and normal tissue and the MAF to SHG index versus depth clearly shows the tissue physiological changes during the process of pulmonary melanoma metastasis. Our results demonstrate that large area NOM succeeds in monitoring the process of pulmonary melanoma metastasis, which can provide a powerful tool for the research in tumor pathophysiology and therapy evaluation.

In this work we demonstrate the potential use of gold nanoparticles as contrast agents for the optical coherence tomography (OCT) imaging technique in dentistry. Here, a new in situ photothermal reduction procedure was developed, producing spherical gold nanoparticles inside dentinal layers and tubules. Gold ions were dispersed in the primer of commercially available dental bonding systems. After the application and permeation in dentin by the modified adhesive systems, the dental bonding materials were photopolymerized concurrently with the formation of gold nanoparticles. The gold nanoparticles were visualized by scanning electron microscopy (SEM). The SEM images show the presence of gold nanospheres in the hybrid layer and dentinal tubules. The diameter of the gold nanoparticles was determined to be in the range of 40 to 120 nm. Optical coherence tomography images were obtained in two- and three-dimensions. The distribution of nanoparticles was analyzed and the extended depth of nanosphere production was determined. The results show that the OCT technique, using in situ formed gold nanoparticles as contrast enhancers, can be used to visualize dentin structures in a non-invasive and non-destructive way.

Discrimination of benign and malignant melanocytic lesions is a major issue in clinical dermatology. Assessment of the thickness of melanoma is critical for prognosis and treatment selection. We aimed to evaluate a novel optical computed tomography (optical-CT) system as a tool for three-dimensional (3-D) imaging of melanocytic lesions and its ability to discriminate benign from malignant melanocytic lesions while simultaneously determining the thickness of invasive melanoma. Seventeen melanocytic lesions, one hemangioma, and normal skin were assessed immediately after their excision by optical-CT and subsequently underwent histopathological examination. Tomographic reconstructions were performed with a back-propagation algorithm calculating a 3-D map of the total attenuation coefficient (AC). There was a statistically significant difference between melanomas, dysplastic nevi, and non-dysplastic nevi, as indicated by Kruskal-Wallis test. Median AC values were higher for melanomas compared with dysplastic and non-dysplastic nevi. No statistically significant difference was observed when thickness values obtained by optical-CT were compared with histological thickness using a Wilcoxon sighed rank test. Our results suggest that optical-CT can be important for the immediate prehistological evaluation of biopsies, assisting the physician for a rapid assessment of malignancy and of the thickness of a melanocytic lesion.

Prostate brachytherapy is a popular prostate cancer treatment option that involves the permanent implantation of radioactive seeds into the prostate. However, contemporary brachytherapy procedure is limited by the lack of an imaging system that can provide real-time seed-position feedback. While many other imaging systems have been proposed, photoacoustic imaging has emerged as a potential ideal modality to address this need, since it could easily be incorporated into the current ultrasound system used in the operating room. We present such a photoacoustic imaging system built around a clinical ultrasound system to achieve the task of visualizing and localizing seeds. We performed several experiments to analyze the effects of various parameters on the appearance of brachytherapy seeds in photoacoustic images. We also imaged multiple seeds in an ex vivo dog prostate phantom to demonstrate the possibility of using this system in a clinical setting. Although still in its infancy, these initial results of a photoacoustic imaging system for the application of prostate brachytherapy seed localization are highly promising.

Ultrasound-modulated optical tomography (UOT) has the potential to reveal optical contrast deep inside soft biological tissues at an ultrasonically determined spatial resolution. The optical imaging depth reported so far has, however, been limited, which prevents this technique from broader applications. Our latest experimental exploration has pushed UOT to an unprecedented imaging depth. We developed and optimized a UOT system employing a photorefractive crystal-based interferometer. A large aperture optical fiber bundle was used to enhance the efficiencies for diffuse light collection and photorefractive two-wave-mixing. Within the safety limits for both laser illumination and ultrasound modulation, the system has attained the ability to image through a tissue-mimicking phantom of 9.4 cm in thickness, which has never been reached previously by UOT.

Photoacoustic imaging, based on ultrasound detected after laser irradiation, is an extension to diagnostic ultrasound for imaging the vasculature, blood oxygenation and the uptake of optical contrast media with promise for cancer diagnosis. For versatile scanning, the irradiation optics is preferably combined with the acoustic probe in an epi-style arrangement avoiding acoustically dense tissue in the acoustic propagation path from tissue irradiation to acoustic detection. Unfortunately epiphotoacoustic imaging suffers from strong clutter, arising from optical absorption in tissue outside the image plane, and from acoustic backscattering. This limits the imaging depth for useful photoacoustic image contrast to typically less than one centimeter. Deformation-compensated averaging (DCA), which takes advantage of clutter decorrelation induced by palpating the tissue with the imaging probe, has previously been proposed for clutter reduction. We demonstrate for the first time that DCA results in reduced clutter in real-time freehand clinical epiphotoacoustic imaging. For this purpose, combined photoacoustic and pulse-echo imaging at 10-Hz frame rate was implemented on a commercial scanner, allowing for ultrasound-based motion tracking inherently coregistered with photoacoustic frames. Results from the forearm and the neck confirm that contrast is improved and imaging depth increased by DCA.

The goal of the study was to evaluate wide-field and high-resolution multimodal optical imaging, including polarization, reflectance, and fluorescence for the intraoperative detection of breast cancer. Lumpectomy specimens were stained with 0.05  mg/ml aqueous solution of methylene blue (MB) and imaged. Wide-field reflectance images were acquired between 390 and 750 nm. Wide-field fluorescence images were excited at 640 nm and registered between 660 and 750 nm. High resolution confocal reflectance and fluorescence images were excited at 642 nm. Confocal fluorescence images were acquired between 670 nm and 710 nm. After imaging, the specimens were processed for hematoxylin and eosin (H&E) histopathology. Histological slides were compared with wide-field and high-resolution optical images to evaluate correlation of tumor boundaries and cellular morphology, respectively. Fluorescence polarization imaging identified the location, size, and shape of the tumor in all the cases investigated. Averaged fluorescence polarization values of tumor were higher as compared to normal tissue. Statistical analysis confirmed the significance of these differences. Fluorescence confocal imaging enabled cellular-level resolution. Evaluation and statistical analysis of MB fluorescence polarization values registered from single tumor and normal cells demonstrated higher fluorescence polarization from cancer. Wide-field high-resolution fluorescence and fluorescence polarization imaging shows promise for intraoperative delineation of breast cancers.

Healthy human colon samples were analyzed ex vivo with a multispectral imaging Mueller polarimeter operating from 500 to 700 nm in a backscattering configuration with diffuse light illumination impinging on the innermost tissue layer, the mucosa. The intensity and polarimetric responses were taken on whole tissues first and after progressive exfoliation of the outer layers afterwards. Moreover, these measurements were carried out with two different substrates (one bright and the other dark) successively placed beneath each sample, allowing a reasonably accurate evaluation of the contributions to the overall backscattered light by the various layers. For the shorter investigated wavelengths (500 to 550 nm) the major contribution comes from mucosa and submucosa, while for the longer wavelengths (650 to 700 nm) muscular tissue and fat also contribute significantly. The depolarization has also been studied and is found to be stronger in the red part of the spectrum, mainly due to the highly depolarizing power of the muscular and fat layers.

A scanning-fiber-based method developed for imaging bioengineered tissue constructs such as synthetic carotid arteries is reported. Our approach is based on directly embedding one or more hollow-core silica fibers within the tissue scaffold to function as micro-imaging channels (MIC). The imaging process is carried out by translating and rotating an angle-polished fiber micro-mirror within the MIC to scan excitation light across the tissue scaffold. The locally emitted fluorescent signals are captured using an electron multiplying CCD camera and then mapped into fluorophore distributions according to fiber micro-mirror positions. Using an optical phantom composed of fluorescent microspheres, tissue scaffolds, and porcine skin, we demonstrated single-cell-level imaging resolution (20 to 30 μm) at an imaging depth that exceeds the photon transport mean free path by one order of magnitude. This result suggests that the imaging depth is no longer constrained by photon scattering, but rather by the requirement that the fluorophore signal overcomes the background "noise" generated by processes such as scaffold autofluorescence. Finally, we demonstrated the compatibility of our imaging method with tissue engineering by visualizing endothelial cells labeled with green fluorescent protein through a ∼ 500  μm thick and highly scattering electrospun scaffold.

Photoacoustic (PA) microscopy (PAM) can image optical absorption contrast with ultrasonic spatial resolution in the optical diffusive regime. Conventionally, accurate quantification in PAM requires knowledge of the optical fluence attenuation, acoustic pressure attenuation, and detection bandwidth. We circumvent this requirement by quantifying the optical absorption coefficients from the acoustic spectra of PA signals acquired at multiple optical wavelengths. With the acoustic spectral method, the absorption coefficients of an oxygenated bovine blood phantom at 560, 565, 570, and 575 nm were quantified with errors of <3%. We also quantified the total hemoglobin concentration and hemoglobin oxygen saturation in a live mouse. Compared with the conventional amplitude method, the acoustic spectral method provides greater quantification accuracy in the optical diffusive regime. The limitations of the acoustic spectral method was also discussed.

The accurate determination of burn depth is critical in the clinical management of burn wounds. Polarization-sensitive optical coherence tomography (PS-OCT) has been proposed as a potentially non-invasive method for determining burn depth by measuring thermally induced changes in the structure and birefringence of skin, and has been investigated in pre-clinical burn studies with animal models and ex vivo human skin. In this study, we applied PS-OCT to the in-vivo imaging of two pediatric burn patients. Deep and superficial burned skins along with contralateral controls were imaged in 3D. The imaging size was 8  mm×6  mm×2  mm in width, length, and depth in the air respectively, and the imaging time was approximately 6 s per volume. Superficially burned skins exhibited the same layered structure as the contralateral controls, but more visible vasculature and reduced birefringence compared to the contralateral controls. In contrast, a deeply burned skin showed loss of the layered structure, almost absent vasculature, and smaller birefringence compared to superficial burns. This study suggested the vasculature and birefringence as parameters for characterizing burn wounds.

This study is designed to test the repeatability of the quantitative analysis of intraretinal layer thickness and cup-disc ratio of the optic nerve head using ultra-high resolution optical coherence tomography (UHR-OCT). Group A, containing 23 eyes of 12 healthy subjects, was imaged twice and group B, containing eight eyes of four subjects, was imaged three times. Intraretinal layers were segmented manually and the cup-to-disc ratio of the optic nerve head was analyzed. Custom-built automatic segmentation software was also used to segment a set of images for comparison. A total of nine intraretinal layers were visualized and extracted manually. With group A, the central foveal thickness was 186.4±15.9  μm (mean±SD). The average retinal thickness was 296.4±21.3  μm. The best repeatability, obtained when two repeated scans were taken, was obtained for the outer nuclear layer followed by the ganglion cell layer, the inner nuclear layer, the retinal nerve fiber layer and the worst was obtained for the outer segment. The intraclass correlation ranged from 0.824 to 0.997. The coefficients of repeatability ranged from 3.24 to 18.3  μm, corresponding to 1.47% to 26.20%. With group B, high interclass correlations were found and the automatic segmentation results were compatible with the manual results. Our results indicated that more retinal features might be imageable using UHR-OCT.

The development of accurate and clinically applicable tools to assess cancer risk is essential to define candidates to undergo screening for early-stage cancers at a curable stage or provide a novel method to monitor chemoprevention treatments. With the use of our recently developed optical technology-spatial-domain low-coherence quantitative phase microscopy (SL-QPM), we have derived a novel optical biomarker characterized by structure-derived optical path length (OPL) properties from the cell nucleus on the standard histology and cytology specimens, which quantifies the nano-structural alterations within the cell nucleus at the nanoscale sensitivity, referred to as nano-morphology marker. The aim of this study is to evaluate the feasibility of the nuclear nano-morphology marker from histologically normal cells, extracted directly from the standard histology specimens, to detect early-stage carcinogenesis, assess cancer risk, and monitor the effect of chemopreventive treatment. We used a well-established mouse model of spontaneous carcinogenesis-ApcMin mice, which develop multiple intestinal adenomas (Min) due to a germline mutation in the adenomatous polyposis coli (Apc) gene. We found that the nuclear nano-morphology marker quantified by OPL detects the development of carcinogenesis from histologically normal intestinal epithelial cells, even at an early pre-adenomatous stage (six weeks). It also exhibits a good temporal correlation with the small intestine that parallels the development of carcinogenesis and cancer risk. To further assess its ability to monitor the efficacy of chemopreventive agents, we used an established chemopreventive agent, sulindac. The nuclear nano-morphology marker is reversed toward normal after a prolonged treatment. Therefore, our proof-of-concept study establishes the feasibility of the SL-QPM derived nuclear nano-morphology marker OPL as a promising, simple and clinically applicable biomarker for cancer risk assessment and evaluation of chemopreventive treatment.

Bioluminescence tomography (BLT) has been successfully applied to the detection and therapeutic evaluation of solid cancers. However, the existing BLT reconstruction algorithms are not accurate enough for cavity cancer detection because of neglecting the void problem. Motivated by the ability of the hybrid radiosity-diffusion model (HRDM) in describing the light propagation in cavity organs, an HRDM-based BLT reconstruction algorithm was provided for the specific problem of cavity cancer detection. HRDM has been applied to optical tomography but is limited to simple and regular geometries because of the complexity in coupling the boundary between the scattering and void region. In the provided algorithm, HRDM was first applied to three-dimensional complicated and irregular geometries and then employed as the forward light transport model to describe the bioluminescent light propagation in tissues. Combining HRDM with the sparse reconstruction strategy, the cavity cancer cells labeled with bioluminescent probes can be more accurately reconstructed. Compared with the diffusion equation based reconstruction algorithm, the essentiality and superiority of the HRDM-based algorithm were demonstrated with simulation, phantom and animal studies. An in vivo gastric cancer-bearing nude mouse experiment was conducted, whose results revealed the ability and feasibility of the HRDM-based algorithm in the biomedical application of gastric cancer detection.

A challenge in photoacoustic tomography (PAT) brain imaging is to compensate for aberrations in the measured photoacoustic data due to their propagation through the skull. By use of information regarding the skull morphology and composition obtained from adjunct x-ray computed tomography image data, we developed a subject-specific imaging model that accounts for such aberrations. A time-reversal-based reconstruction algorithm was employed with this model for image reconstruction. The image reconstruction methodology was evaluated in experimental studies involving phantoms and monkey heads. The results establish that our reconstruction methodology can effectively compensate for skull-induced acoustic aberrations and improve image fidelity in transcranial PAT.

The advent of molecularly targeted therapies requires effective identification of the various cell types of non-small cell lung carcinomas (NSCLC). Currently, cell type diagnosis is performed using small biopsies or cytology specimens that are often insufficient for molecular testing after morphologic analysis. Thus, the ability to rapidly recognize different cancer cell types, with minimal tissue consumption, would accelerate diagnosis and preserve tissue samples for subsequent molecular testing in targeted therapy. We report a label-free molecular vibrational imaging framework enabling three-dimensional (3-D) image acquisition and quantitative analysis of cellular structures for identification of NSCLC cell types. This diagnostic imaging system employs superpixel-based 3-D nuclear segmentation for extracting such disease-related features as nuclear shape, volume, and cell-cell distance. These features are used to characterize cancer cell types using machine learning. Using fresh unstained tissue samples derived from cell lines grown in a mouse model, the platform showed greater than 97% accuracy for diagnosis of NSCLC cell types within a few minutes. As an adjunct to subsequent histology tests, our novel system would allow fast delineation of cancer cell types with minimum tissue consumption, potentially facilitating on-the-spot diagnosis, while preserving specimens for additional tests. Furthermore, 3-D measurements of cellular structure permit evaluation closer to the native state of cells, creating an alternative to traditional 2-D histology specimen evaluation, potentially increasing accuracy in diagnosing cell type of lung carcinomas.

For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4× histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

Near-infrared imaging of lymphatic drainage of injected indocyanine green (ICG) has emerged as a new technology for clinical imaging of lymphatic architecture and quantification of vessel function, yet the imaging capabilities of this approach have yet to be quantitatively characterized. We seek to quantify its capabilities as a diagnostic tool for lymphatic disease. Imaging is performed in a tissue phantom for sensitivity analysis and in hairless rats for in vivo testing. To demonstrate the efficacy of this imaging approach to quantifying immediate functional changes in lymphatics, we investigate the effects of a topically applied nitric oxide (NO) donor glyceryl trinitrate ointment. Premixing ICG with albumin induces greater fluorescence intensity, with the ideal concentration being 150  μg/mL ICG and 60  g/L albumin. ICG fluorescence can be detected at a concentration of 150  μg/mL as deep as 6 mm with our system, but spatial resolution deteriorates below 3 mm, skewing measurements of vessel geometry. NO treatment slows lymphatic transport, which is reflected in increased transport time, reduced packet frequency, reduced packet velocity, and reduced effective contraction length. NIR imaging may be an alternative to invasive procedures measuring lymphatic function in vivo in real time.

We have developed spectrally encoded photoacoustic microscopy using a digital mirror device for multi-wavelength tomography, which enables fast spectral imaging of optical absorption. The optical illumination wavelengths are multiplexed at a laser pulse repetition rate of ∼ 2  kHz. Liquid samples, whole blood, and blood vessels in mouse ears were imaged. Compared with internal wavelength tuning of a narrow-band laser, external wavelength tuning based on a digital mirror device improves the data acquisition speed of spectral photoacoustic microscopy. Compared with external wavelength scanning of a wide-band laser with the same pulse energy, spectral encoding improves the signal-to-noise ratio.

Axial epi-illuminating light transmitting a >1.3-numerical-aperture microscope objective creates an excitation volume at focus with size and shape dictated by diffraction and due to refraction by the objective and by the coverslip interface separating a specimen in aqueous buffer from the oil immersion objective. The evanescent field on the coverslip aqueous side affects primarily the excitation volume axial dimension as the specimen in focus approaches the interface to within a few hundred nanometers. Following excitation, an excited stationary dipole moment emits fluorescence in a spatially varying pattern collected over the large objective aperture. Collected light propagates in parallel rays toward the tube lens that forms a real three-dimensional image that is decoded to identify dipole orientation. An integral representation of the excitation and emitted fields for infinity-corrected optics-including effects of finite conjugate illumination, fluorescence emission near an interface, emitter dipole orientation, spherical aberration, light transmission through a dichroic filter, and for real microscopic specifications-accurately models observed field intensities including the substantial excitation from the evanescent field. The goal is to develop and verify the practical depiction of excitation and emission in a real microscope for quantitative interpretation of the 3-D emission pattern.

The strong optical scattering of skin tissue makes it very difficult for optical coherence tomography (OCT) to achieve deep imaging in skin. Significant optical clearing of in vivo rat skin sites was achieved within 15 min by topical application of an optical clearing agent PEG-400, a chemical enhancer (thiazone or propanediol), and physical massage. Only when all three components were applied together could a 15 min treatment achieve a three fold increase in the OCT reflectance from a 300 μm depth and 31% enhancement in image depth Zthreshold.

In vivo measurement of photosensitizer concentrations may optimize clinical photodynamic therapy (PDT). Fluorescence differential path-length spectroscopy (FDPS) is a non-invasive optical technique that has been shown to accurately quantify the concentration of Foscan® in rat liver. As a next step towards clinical translation, the effect of two liposomal formulations of mTHPC, Fospeg® and Foslip®, on FDPS response was investigated. Furthermore, FDPS was evaluated in target organs for head-and-neck PDT. Fifty-four healthy rats were intravenously injected with one of the three formulations of mTHPC at 0.15  mg kg−1. FDPS was performed on liver, tongue, and lip. The mTHPC concentrations estimated using FDPS were correlated with the results of the subsequent harvested and chemically extracted organs. An excellent goodness of fit (R2) between FDPS and extraction was found for all formulations in the liver (R2 = 0.79). A much lower R2 between FDPS and extraction was found in lip (R2 = 0.46) and tongue (R2 = 0.10). The lower performance in lip and in particular tongue was mainly attributed to the more layered anatomical structure, which influences scattering properties and photosensitizer distribution.

Fluorescence spectroscopy has been widely investigated as a technique for identifying pathological tissue; however, unrelated subject-to-subject variations in spectra complicate data analysis and interpretation. We describe and evaluate a new biosensing technique, differential laser-induced perturbation spectroscopy (DLIPS), based on deep ultraviolet (UV) photochemical perturbation in combination with difference spectroscopy. This technique combines sequential fluorescence probing (pre- and post-perturbation) with sub-ablative UV perturbation and difference spectroscopy to provide a new spectral dimension, facilitating two improvements over fluorescence spectroscopy. First, the differential technique eliminates significant variations in absolute fluorescence response within subject populations. Second, UV perturbations alter the extracellular matrix (ECM), directly coupling the DLIPS response to the biological structure. Improved biosensing with DLIPS is demonstrated in vivo in a murine model of chemically induced skin lesion development. Component loading analysis of the data indicates that the DLIPS technique couples to structural proteins in the ECM. Analysis of variance shows that DLIPS has a significant response to emerging pathology as opposed to other population differences. An optimal likelihood ratio classifier for the DLIPS dataset shows that this technique holds promise for improved diagnosis of epithelial pathology. Results further indicate that DLIPS may improve diagnosis of tissue by augmenting fluorescence spectra (i.e. orthogonal sensing).

Studies with circulating ribonucleic acid (RNA) not only provide new targets for cancer detection, but also open up the possibility of noninvasive gene expression profiling for cancer. In this paper, we developed a surface-enhanced Raman scattering (SERS), platform for detection and differentiation of serum RNAs of colorectal cancer. A novel three-dimensional (3-D), Ag nanofilm formed by dry MgSO4 aggregated silver nanoparticles, Ag NP, as the SERS-active substrate was presented to effectively enhance the RNA Raman signals. SERS measurements were performed on two groups of serum RNA samples. One group from patients, n = 55 with pathologically diagnosed colorectal cancer and the other group from healthy controls, n = 45. Tentative assignments of the Raman bands in the normalized SERS spectra demonstrated that there are differential expressions of cancer-related RNAs between the two groups. Linear discriminate analysis, based on principal component analysis, generated features can differentiate the colorectal cancer SERS spectra from normal SERS spectra with sensitivity of 89.1 percent and specificity of 95.6 percent. This exploratory study demonstrated great potential for developing serum RNA SERS analysis into a useful clinical tool for label-free, noninvasive screening and detection of colorectal cancers.

Accurate determination of the optical properties (the absorption coefficient μa and the reduced scattering coefficient μs′) of tissues is very important in a variety of diagnostic and therapeutic procedures. Optical diffusion theory is frequently used as the forward model for describing the photon transfer in media with large reduced albedos (a′) and in large source-detector separations (SDS). Several other methods (PN approximation, hybrid diffusion-P3 approximation) have also been published that describe photon transfer in media with low a′ or small SDSs. We studied the theoretical models for the steady-state spatially resolved diffuse reflectance measurement to accurately determine μa and μs′ at large a′ range but small SDSs. Instead of using a single model, a joint derivation method is proposed. The developed method uses one of the best aforementioned theoretical methods separately in five ranges of a′ determined from several forward models. In the region of small SDSs (the range between 0.4 and 8 mm) and large a′ range (between 0.5 and 0.99), the best theoretical derivation model was determined. The results indicate that the joint derivation method can improve the derivation accuracy and that a′ range can be determined by the steady-state spatially resolved diffuse reflectance measurement.

An on-chip system is presented with integrated architectures for digital microfluidic actuation and sensing. Localized actuation is brought about by a digital microfluidic multiplexer layout that overcomes the challenges of multi-microdrop interference, and complete two-dimensional motion is shown for microdrops on a 14×14 grid with minimized complexity by way of 14+14 inputs. At the same time, microdrop sensing is demonstrated in a folded-cavity design for enhanced optical intensity probing of internal fluid refractive indices. The heightened intensities from this on-chip refractometer are shown to have a linear response to the underlying fluid refractive index. An electro-dispensing technique is used to fabricate the folded-cavity optical architecture in a format that is tuned for the desired refractive index range and sensitivity. The overall lab-on-a-chip system is successful in integrating localized microdrop actuation and sensing.

We present a device and method for performing vector transmission spectroscopy on biological specimens at terahertz (THz) frequencies. The device consists of artificial dielectric birefringence obtained from silicon microfluidic grating structures. The device can measure the complex dielectric function of a liquid, across a wide THz band of 2 to 5.5 THz, using a Fourier transform infrared spectrometer. Measurement data from a range of liquid specimens, including sucrose, salmon deoxyribonucleic acid (DNA), herring DNA, and bovine serum albumin protein solution in water are presented. The specimen handling is simple, using a microfluidic channel. The transmission through the device is improved significantly and thus the measurement accuracy and bandwidth are increased.

We present a new approach to the analysis of radiance in turbid media. The approach combines data from spectral, angular and spatial domains in a form of spectro-angular maps. Mapping provides a unique way to visualize details of light distribution in turbid media and allows tracking changes with distance. Information content of experimental spectro-angular maps is verified by a direct comparison with simulated data when an analytical solution of the radiative transfer equation is used. The findings deepen our understanding of the light distribution in a homogenous turbid medium and provide a first step toward applying the spectro-angular mapping as a diagnostic tool for tissue characterization.

Assessing noninvasively cerebral autoregulation, the protective mechanism of the brain to maintain constant cerebral blood flow despite changes in blood pressure, is challenging. Infants on life support system (ECMO) for cardiorespiratory failure are at risk of cerebral autoregulation impairment and consequent neurological problems. We measured oxyhaemoglobin concentration (HbO2) by multichannel (12 channels) near-infrared spectroscopy (NIRS) in six infants during sequential changes in ECMO flow. Wavelet cross-correlation (WCC) between mean arterial pressure (MAP) and HbO2 was used to construct a time-frequency representation of the concordance between the two signals to assess the nonstationary aspect of cerebral autoregulation and investigate regional variations. Group data showed that WCC increases with decreasing ECMO flow indicating higher concordance between MAP and HbO2 and demonstrating loss of cerebral autoregulation at low ECMO flows. Statistically significant differences in WCC were observed between channels placed on the right and left scalp with channels on the right exhibiting higher values of WCC suggesting that the right hemisphere was more susceptible to disruption of cerebral autoregulation. Multichannel NIRS in conjunction with wavelet analysis methods can be used to assess regional variations in dynamic cerebral autoregulation with important clinical application in the management of critically ill children on life support systems.

As nanotechnology continues to develop, an assessment of nanoparticles' toxicity becomes very crucial for biomedical applications. The current study examines the deleterious effects of pre-irradiated gold nanoparticles (GNPs) solutions on primary rat kidney cells (PRKCs). Spectroscopic and transmission electron microscopic studies demonstrated that exposure of 15 nm GNPs in size to pulsed laser caused a reduction both in optical density and mean particle diameter. GNPs showed an aggregation when added to the cell culture medium (DMEM). This aggregation was markedly decreased upon adding serum to the medium. Under our experimental conditions, trypan blue and MTT assays revealed no significant changes in cell viability when PRKCs were incubated with non-irradiated GNPs over a period of 72 h and up to 4 nM GNPs concentration. On the contrary, when cells were incubated with irradiated GNPs a significant reduction in PRKCs viability was revealed.

We investigated the effect of low-level laser radiation on rescuing hair cells of the cochlea after acute acoustic trauma and hearing loss. Nine rats were exposed to noise. Starting the following day, the left ears (NL ears) of the rats were irradiated at an energy output of 100 to 165  mW/cm2 for 60 min for 12 days in a row. The right ears (N ears) were considered as the control group. Frequency-specific hearing levels were measured before the noise exposure and also after the 1st, 3rd to 5th, 8th to 10th and 12th irradiations. After the 12th treatment, hair cells were observed using a scanning electron microscope. Compared to initial hearing levels at all frequencies, thresholds increased markedly after noise exposure. After the 12th irradiation, hearing threshold was significantly lower for the NL ears compared to the N ears. When observed using an electron microscope, the number of hair cells in the middle turn of the NL ears was significantly larger than that of the N ears. Our findings suggest that low-level laser irradiation promotes recovery of hearing thresholds after acute acoustic trauma.

To expand the optional laser wavelengths of photodynamic therapy (PDT) for port wine stain (PWS), the feasibility of applying a 457 nm laser to the PDT for infantile PWS was analyzed by mathematical simulation and was validated by clinical experiment. Singlet oxygen yield of 457 nm PDT or 532 nm PDT in an infantile PWS model and an adult PWS model was theoretically simulated. Fifteen PWS patients (14 infants and 1 adult) with 40 spots were treated with 457 nm (20 spots) and 532 nm (20 spots), respectively, in two PDT courses. Simulation results showed that under the same power density and irradiation time, singlet oxygen yield of 457 nm PDT and 532 nm PDT are similar in infantile PWS vessels. Yet, in adult PWS vessels, singlet oxygen yield of 457 nm PDT is lower than 532 nm PDT. Clinical outcomes showed that no statistic difference existed between 457 nm PDT and 532 nm PDT for infantile PWS. The result of this study suggested that 457 nm wavelength laser has the potential to be applied in PDT for infantile PWS.

Ophthalmic surgery may benefit from use of more precise fiber delivery systems during laser surgery. Some current ophthalmic surgical techniques rely on tedious mechanical dissection of tissue layers. In this study, chains of sapphire microspheres integrated into a hollow waveguide distal tip are used for erbium:YAG laser ablation studies in contact mode with ophthalmic tissues, ex vivo. The laser's short optical penetration depth combined with the small spot diameters achieved with this fiber probe may provide more precise tissue removal. One-, three-, and five-microsphere chain structures were characterized, resulting in FWHM diameters of 67, 32, and 30 μm in air, respectively, with beam profiles comparable to simulations. Single Er:YAG pulses of 0.1 mJ and 75-μs duration produced ablation craters with average diameters of 44, 30, and 17 μm and depths of 26, 10, and 8 μm, for one-, three-, and five-sphere structures, respectively. Microsphere chains produced spatial filtering of the multimode Er:YAG laser beam and fiber, providing spot diameters not otherwise available with conventional fiber systems. Because of the extremely shallow treatment depth, compact focused beam, and contact mode operation, this probe may have potential for use in dissecting epiretinal membranes and other ophthalmic tissues without damaging adjacent retinal tissue.

Laparoscopic/robotic partial nephrectomy (LPN) is increasingly considered for small renal tumors (RT). This demands new compatible surgical tools for RT-resection, such as lasers, to optimize cutting and coagulation. This work aims to characterize ex vivo handling requirements for six medically approved laser devices emitting different light wavelengths (940, 1064, 1318, 1470, 1940, and 2010 nm) amenable for LPN. Incisions were made by laser fibers driven by a computer-controlled stepping motor allowing precise linear movement with a preset velocity at a fixed fiber-tip distance to tissue. Optical parameters were measured on 200 μm tissue slices. Cutting quality depended on power output, fiber velocity and fiber-tip distance to tissue. Contact manner is suitable for cutting while a noncontact manner (5 mm distance) induces coagulation. Ablation threshold differs for each wavelength. Ablation depth is proportional to power output (within limit) while axial and superficial coagulation remains mostly constant. Increased fiber velocity compromises the coagulation quality. Optical parameters of porcine kidney tissue demonstrate that renal absorption coefficient follows water absorption in the 2 μm region while for other spectral regions (900 to 1500 and 1 μm) the tissue effects are influenced by other chromophores and scattering. Tissue color changes demonstrate dependencies on irradiance, scan velocity, and wavelength. Current results clearly demonstrate that surgeons considering laser-assisted RT excisions should be aware of the mentioned technical parameters (power output, fiber velocity and fiber-tip tissue-distance) rather than wavelength only.