Real‐time optical spectroscopic monitoring of nonirrigated lesion progression within atrial and ventricular tissues

Despite considerable advances in guidance of radiofrequency ablation (RFA) therapy for the treatment of cardiac arrhythmias, success rates have been hampered by a lack of tools for precise intraoperative evaluation of lesion extent. Near‐infrared spectroscopic (NIRS) techniques are sensitive to tissue structural and biomolecular properties, characteristics that are directly altered by radiofrequency (RF) treatment. In this work, a combined NIRS‐RFA catheter is developed for real‐time monitoring of tissue reflectance during RF energy delivery. An algorithm is proposed for processing NIR spectra to approximate nonirrigated lesion depth in both atrial and ventricular tissues. The probe optical geometry was designed to bias measurement influence toward absorption enabling enhanced sensitivity to changes in tissue composition. A set of parameters termed “lesion optical indices” are defined encapsulating spectral differences between ablated and unablated tissue. Utilizing these features, a model for real‐time tissue spectra classification and lesion size estimation is presented. Experimental validation conducted within freshly excised porcine cardiac specimens showed strong concordance between algorithm estimates and post‐hoc tissue assessment.      

Investigation of the source‐detector separation in near infrared spectroscopy for healthy and clinical applications

Understanding near infrared light propagation in tissue is vital for designing next generation optical brain imaging devices. Monte Carlo (MC) simulations provide a controlled mechanism to characterize and evaluate contributions of diverse near infrared spectroscopy (NIRS) sensor configurations and parameters. In this study, we developed a multilayer adult digital head model under both healthy and clinical settings and assessed light‐tissue interaction through MC simulations in terms of partial differential pathlength, mean total optical pathlength, diffuse reflectance, detector light intensity and spatial sensitivity profile of optical measurements. The model incorporated four layers: scalp, skull, cerebrospinal‐fluid and cerebral cortex with and without a customizable lesion for modeling hematoma of different sizes and depths. The effect of source‐detector separation (SDS) on optical measurements' sensitivity to brain tissue was investigated. Results from 1330 separate simulations [(4 lesion volumes × 4 lesion depths for clinical +3 healthy settings) × 7 SDS × 10 simulation = 1330)] each with 100 million photons indicated that selection of SDS is critical to acquire optimal measurements from the brain and recommended SDS to be 25 to 35 mm depending on the wavelengths to obtain optical monitoring of the adult brain function. The findings here can guide the design of future NIRS probes for functional neuroimaging and clinical diagnostic systems.      

Noninvasive and sensitive optical assessment of brain death

Brain death is an irreversible loss of all brain functions, and the assessment is crucial for organ supply for transplantation. The noninvasive, sensitive, universally available and timely ancillary method to assess brain death has not been established. Here, we attempted to explore a noninvasive way in brain death assessment. Eighteen brain‐dead patients and 20 healthy subjects were measured by near‐infrared spectroscopy (NIRS), with a multiple‐phase protocol at varied fraction of inspired O₂ (FIO₂). We found that the concentration changes ratios of oxyhemoglobin to deoxyhemoglobin (Δ[HbO₂]/Δ[Hb]) in the cerebral cortex of brain‐dead patients were significantly higher than those of healthy subjects. And, the Δ[HbO₂]/Δ[Hb] in low‐to‐high FIO₂ phase was most sensitive to distinguish brain‐dead patients from healthy subjects, with a recommended threshold ranged in 1.40~1.50. The innovative incorporation of NIRS and a varied FIO₂ protocol was shown to be a noninvasive and reliable way in assessing brain death. This successful attempt of NIRS application is a help for fast and accurate evaluation of brain death, promptly offering quality‐assured donor organs and indicate us a protocol‐aided way to expand the use of NIRS.      

Correlation between tissue oxygenation and erythrocytes elasticity

In this paper, near‐infrared spectroscopy (NIRS) and jumping optical tweezers were used to measure the tissue oxygenation and the elasticity of erythrocytes, respectively. The correlation between tissue oxygenation induced by arterial occlusion test (AOT) and the mechanical properties of individual erythrocytes from a blood sample obtained after AOT was studied. The experimental results show a linear correlation between the oxygenation signal caused by AOT and the elasticity of erythrocytes. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Spectral correction for handheld optoacoustic imaging by means of near‐infrared optical tomography in reflection mode

In vivo imaging of tissue/vasculature oxygen saturation levels is of prime interest in many clinical applications. To this end, the feasibility of combining two distinct and complementary imaging modalities is investigated: optoacoustics (OA) and near‐infrared optical tomography (NIROT), both operating noninvasively in reflection mode. Experiments were conducted on two optically heterogeneous phantoms mimicking tissue before and after the occurrence of a perturbation. OA imaging was used to resolve submillimetric vessel‐like optical absorbers at depths up to 25 mm, but with a spectral distortion in the OA signals. NIROT measurements were utilized to image perturbations in the background and to estimate the light fluence inside the phantoms at the wavelength pair (760 nm, 830 nm). This enabled the spectral correction of the vessel‐like absorbers' OA signals: the error in the ratio of the absorption coefficient at 830 nm to that at 760 nm was reduced from 60%‐150% to 10%‐20%. The results suggest that oxygen saturation (SO ₂) levels in arteries can be determined with <10% error and furthermore, that relative changes in vessels' SO ₂ can be monitored with even better accuracy. The outcome relies on a proper identification of the OA signals emanating from the studied vessels.      

Moving tissue spectral window to the deep‐ultraviolet via optical clearing

The optical immersion clearing technique has been successfully applied through the last 30 years in the visible to near infrared spectral range, and has proven to be a promising method to promote the application of optical technologies in clinical practice. To investigate its potential in the ultraviolet range, collimated transmittance spectra from 200 to 1000 nm were measured from colorectal muscle samples under treatment with glycerol‐water solutions. The treatments created two new optical windows with transmittance efficiency peaks at 230 and 300 nm, with magnitude increasing with glycerol concentration in the treating solution. Such discovery opens the opportunity to develop clinical procedures to perform diagnosis or treatments in the ultraviolet.     

Multiparameter wide‐field integrated optical imaging system‐based spatially modulated illumination and laser speckles in model of tissue injuries

In this report, an integrated optical platform based on spatial illumination together with laser speckle contrast technique was utilized to measure multiple parameters in live tissue including absorption, scattering, saturation, composition, metabolism, and blood flow. Measurements in three models of tissue injury including drug toxicity, artery occlusion, and acute hyperglycemia were used to test the efficacy of this system. With this hybrid apparatus, a series of structured light patterns at low and high spatial frequencies are projected onto the tissue surface and diffuse reflected light is captured by a CCD camera. A six position filter wheel, equipped with four bandpass filters centered at wavelengths of 650, 690, 800 and 880 nm is placed in front of the camera. Then, light patterns are blocked and a laser source at 650 nm illuminates the tissue while the diffusely reflected light is captured by the camera through the two remaining open holes in the wheel. In this manner, near‐infrared (NIR) and laser speckle images are captured and stored together in the computer for off‐line processing to reconstruct the tissue's properties. Spatial patterns are used to differentiate the effects of tissue scattering from those of absorption, allowing accurate quantification of tissue hemodynamics and morphology, while a coherent light source is used to study blood flow changes, a feature which cannot be measured with the NIR structured light. This combined configuration utilizes the strengths of each system in a complementary way, thus collecting a larger range of sample properties. In addition, once the flow and hemodynamics are measured, tissue oxygen metabolism can be calculated, a property which cannot be measured independently. Therefore, this merged platform can be considered a multiparameter wide‐field imaging and spectroscopy modality. Overall, experiments demonstrate the capability of this spatially coregistered imaging setup to provide complementary, useful information of various tissue metrics in a simple and noncontact manner, making it attractive for use in a variety of biomedical applications.     

Muscle oxygenation dynamics in response to electrical stimulation as measured with near‐infrared spectroscopy: A pilot study

Neuromuscular electrical stimulation (NMES) is used for preventing muscle atrophy and improving muscle strength in patients and healthy people. However, the current intensity of NMES is usually set at a level that causes the stimulated muscles to contract. This typically causes pain. Quantifying the instantaneous changes in muscle microcirculation and metabolism during NMES before muscle contraction occurs is crucial, because it enables the current intensity to be optimally tuned, thereby reducing the NMES‐induced muscle pain and fatigue. We applied near‐infrared spectroscopy (NIRS) to measure instantaneous tissue oxygenation and deoxygenation changes in 43 healthy young adults during NMES at 10, 15, 20, 25, 30, and 35 mA. Having been stabilized at the NIRS signal baseline, the tissue oxygenation and total hemoglobin concentration increased immediately after stimulation in a dose‐dependent manner (P < 0.05) until stimulation was stopped at the level causing muscle contraction without pain. Tissue deoxygenation appeared relatively unchanged during NMES. We conclude that NIRS can be used to determine the optimal NMES current intensity by monitoring oxygenation changes.      

The hemodynamic changes during cupping therapy monitored by using an optical sensor embedded cup

Cupping therapy is one form of alternative medicine that is used widely across the world. Although the applications of cupping therapy including pain relief have a 1000‐year history, the therapeutic effect of cupping is still questionable due to a lack of scientific evidence. Therefore, in the present study, we embedded a near‐infrared spectroscopic sensor into a suction cup to monitor the hemodynamic changes on the treated site while the hemodynamics at the surrounding tissue of the cup was also simultaneously monitored by another near‐infrared spectroscopic sensor. The results from 10 healthy male subjects show a dramatic increase of the oxy‐hemoglobin (OHb) and deoxy‐hemoglobin (RHb) concentrations at the treatment site while the OHb and RHb levels were decreased at the surrounding tissue. Moreover, after the treatment, we observed that the OHb concentrations were maintained at a higher level than before treatment at both sites, which may demonstrate how cupping therapy works for treatment. In summary, the results showed that cupping therapy increases blood volume and tissue oxygenation at the treatment site while those were slightly decreased at the surrounding tissue. This study showed that the embedding of near‐infrared spectroscopy in a cupping system could offer a better understanding of the mechanism of cupping therapy.      

Comparison of pulsed photothermal radiometry,optical coherence tomography and ultrasound for melanoma thickness measurement in PDMS tissue phantoms

Melanoma accounts for 75% of all skin cancer deaths. Pulsed photothermal radiometry (PPTR), optical coherence tomography (OCT) and ultrasound (US) are non‐invasive imaging techniques that may be used to measure melanoma thickness, thus, determining surgical margins. We constructed a series of PDMS tissue phantoms simulating melanomas of different thicknesses. PPTR, OCT and US measurements were recorded from PDMS tissue phantoms and results were compared in terms of axial imaging range, axial resolution and imaging time. A Monte Carlo simulation and three‐dimensional heat transfer model was constructed to simulate PPTR measurement. Experimental results show that PPTR and US can provide a wide axial imaging range (75 μm–1.7 mm and 120–910 μm respectively) but poor axial resolution (75 and 120 μm respectively) in PDMS tissue phantoms, while OCT has the most superficial axial imaging range (14–450 μm) but highest axial resolution (14 μm). The Monte Carlo simulation and three‐dimensional heat transfer model give good agreement with PPTR measurement. PPTR and US are suited to measure thicker melanoma lesions (<$>><$>400 μm), while OCT is better to measure thin melanoma lesions (<$><<$>400 μm). (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Optical properties of mice's stool in 550 to 1000 nm wavelength range

The aim of this work was to measure optical properties of stool of mice to provide this relevant wavelength‐dependent behavior for optical imaging modalities such as fluorescent molecular tomography and near‐infrared optical tomography. BALB/c nude female mice were studied and optical properties of the stool were determined by employing the inverse adding‐doubling approach. The animals were kept on chlorophyll‐free diet. Nine stool samples were measured. The wavelength‐dependent behavior of absorption and scattering in 550 to 1000 nm range is presented. The reduced scattering spectrum is fitted to the Mie scattering approximation in the near‐infrared (NIR) wavelength range and to the Mie + Rayleigh approximation in visible/NIR range with the fitting coefficients presented. The study revealed that the absorption spectrum of stool can lead to crosstalk with the spectrum of hemoglobin in the NIR range.      

Quantitative multi‐spectral oxygen saturation measurements independent of tissue optical properties

Imaging of tissue oxygenation is important in several applications associated with patient care. Optical sensing is commonly applied for assessing oxygen saturation but is often restricted to local measurements or else it requires spectral and spatial information at the expense of time. Many methods proposed so far require assumptions on the properties of measured tissue. In this study we investigated a computational method that uses only multispectral information and quantitatively computes tissue oxygen saturation independently of tissue optical properties. The method is based on linear transformations of measurements in three isosbestic points. We investigated the ideal isosbestic point combination out of six isosbestic points available for measurement in the visible and near‐infrared region that enable accurate oxygen saturation computation. We demonstrate this method on controlled tissue mimicking phantoms having different optical properties and validated the measurements using a gas analyzer. A mean error of 2.9 ± 2.8% O₂Sat was achieved. Finally, we performed pilot studies in tissues in‐vivo by measuring dynamic changes in fingers subjected to vascular occlusion, the vasculature of mouse ears and exposed mouse organs.

Selected steps of spectral transformations applied to oxygenation spectra. The original reflectance spectrum M(λ) is transformed in step 1 to overlap with reference spectra (grey) in three isosbestic points, resulting in M″(λ). In step 2, the gradient of M″(λ) is computed resulting in M″_grad(λ), which can be used for quantitative oxygenation computation.     

Second harmonic imaging of plants tissues and cell implosion using two‐photon process in ZnO nanoparticles

The optical properties of colloidal ZnO nanoparticle (NP) solutions, with size ranging from several nm to around 200 nm, have been tailored to have high optical nonlinearity for bioimaging with no auto‐fluorescence above 750 nm and minimal auto‐fluorescence below 750 nm. The high second harmonic conversion efficiency enables selective tissue imaging and cell tracking using tunable near‐infrared femtosecond laser source ranging from 750‐980 nm. For laser energies exceeding the two‐photon energy of the bandgap of ZnO (half of 3.34 eV), the SHG signal greatly decreases and the two‐photon emission becomes the dominant signal. The heat generated due to two‐photon absorption within the ZnO NPs enable selective cell or localized tissue destruction using excitation wavelength ranging from 710–750 nm. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

General model for depth‐resolved estimation of the optical attenuation coefficients in optical coherence tomography

We present the proof of concept of a general model that uses the tissue sample transmittance as input to estimate the depth‐resolved attenuation coefficient of tissue samples using optical coherence tomography (OCT). This method allows us to obtain an image of tissue optical properties instead of intensity contrast, guiding diagnosis and tissues differentiation, extending its application from thick to thin samples. The performance of our method was simulated and tested with the assistance of a home built single‐layered and multilayered phantoms (~100 μm each layer) with known attenuation coefficient on the range of 0.9 to 2.32 mm^?1. It is shown that the estimated depth‐resolved attenuation coefficient recovers the reference values, measured by using an integrating sphere followed by the inverse adding doubling processing technique. That was corroborated for all situations when the correct transmittance value is used with an average difference of 7%. Finally, we applied the proposed method to estimate the depth‐resolved attenuation coefficient for a thin biological sample, demonstrating the ability of our method on real OCT images.     

Hyperspectral optical coherence tomography for in vivo visualization of melanin in the retinal pigment epithelium

Previous studies for melanin visualization in the retinal pigment epithelium (RPE) have exploited either its absorption properties (using photoacoustic tomography or photothermal optical coherence tomography [OCT]) or its depolarization properties (using polarization sensitive OCT). However, these methods are only suitable when the melanin concentration is sufficiently high. In this work, we present the concept of hyperspectral OCT for melanin visualization in the RPE when the concentration is low. Based on white light OCT, a hyperspectral stack of 27 wavelengths (440‐700 nm) was created in post‐processing for each depth‐resolved image. Owing to the size and shape of the melanin granules in the RPE, the variations in backscattering coefficient as a function of wavelength could be identified—a result which is to be expected from Mie theory. This effect was successfully identified both in eumelanin‐containing phantoms and in vivo in the low‐concentration Brown Norway rat RPE.      

Fluorescent styryl dyes applied as fast optical probes of cardiac action potential

Several styryl dyes were tested as fast optical probes of membrane action potentials in mammalian heart muscle tissue. After staining, atrial specimens were superfused in physiological salt solution, and fluorescence was excited by an argon ion laser. Excitation spot size on the surface of the preparation was 60 m in diameter. Dyes RH 160, RH 237, and RH 421 performed excellently as fast fluorescent probes of cardiac membrane potential. Fractional fluorescence changes, F/F, due to the action potential were in the range 2 to 6% at 514.5 nm excitation. Rise times of the action potential onset detected with each of the dyes were less than 0.5 ms, which is as fast or even faster than microelectrode measurements (atria of the rat). Thus membrane potential changes could be monitored with high resolution in both time and space. Emission spectra from heart muscle preparations stained with these dyes were shifted to shorter wavelengths by 70 nm and more as compared to spectra of the dyes in ethanol solution. The fluorescence spectrum of RH 160 at resting potential and the spectrum recorded during the plateau phases of the action potential were measured and showed no difference within the spectral resolution. As can be concluded from measurements of fluorescence changes at different excitation wavelengths, electrochromism cannot be the only mechanism causing the potential response.     

405 nm versus 633 nm for protoporphyrin IX excitation in fluorescence‐guided stereotactic biopsy of brain tumors

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5‐aminolevulinic‐acid‐induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50‐fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well‐defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose‐gel phantoms. Even in direct contact, blood layers of 20–40 µm between probe and tissue must be expected, obscuring 405‐nm‐excited PpIX fluorescence almost completely, but reducing the 633‐nm‐excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX‐assisted detection of high‐grade gliomas in stereotactic biopsy.

PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation.     

New near-infrared optical probes of cardiac electrical activity

Styryl voltage-sensitive dyes (e.g., di-4-ANEPPS) have been widely and successfully used as probes for mapping membrane potential changes in cardiac cells and tissues. However, their utility has been somewhat limited because their excitation wavelengths have been restricted to the 450- to 550-nm range. Longer excitation/emission wavelength probes can minimize interference from endogenous chromophores and, because of decreased light scattering and lower absorption by endogenous chromophores, improve recording from deeper tissue layers. In this article, we report efforts to develop new potentiometric styryl dyes that have excitation wavelengths ranging above 700 nm and emission spectra extending to 900 nm. Three dyes for cardiac optical mapping were investigated in depth from several hundred dyes containing 47 variants of the styryl chromophores. Absorbance and emission spectra in ethanol and multilamellar vesicles, as well as voltage-dependent spectral changes in a model lipid bilayer, have been recorded for these dyes. Optical action potentials were recorded in typical cardiac tissues (rat, guinea pig, pig) and compared with those of di-4-ANEPPS. The voltage sensitivities of the fluorescence of these new potentiometric indicators are as good as those of the widely used ANEP series of probes. In addition, because of molecular engineering of the chromophore, the new dyes provide a wide range of dye loading and washout time constants. These dyes will enable a series of new experiments requiring the optical probing of thick and/or blood-perfused cardiac tissues.     

Rapid intra‐operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears

Herein, a technique to analyze air‐dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR‐IR) spectroscopy is presented. Spectral tumor markers—absorption bands of glycogen—are identified in the ATR‐IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo‐sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze‐thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real‐time intra‐operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.      

Gender differences in brain networks during verbal Sternberg tasks: A simultaneous near‐infrared spectroscopy and electro‐encephalography study

Gender differences in psychological processes have been of great interest in a variety of fields including verbal fluency, emotion processing and working memory. Previous studies suggested that women outperform men in verbal working memory (VWM). However, the inherent mechanisms are still unclear. To obtain a deeper insight into the gender differences in brain networks in VWM, this study used near‐infrared spectroscopy (NIRS) and electro‐encephalography (EEG) simultaneously to investigate gender‐related brain networks during verbal Sternberg tasks. NIRS results confirmed that women surpass men in VWM from the perspective of both brain activation and connectivity. Results of EEG (effective connectivity and event‐related spectral power) showed that men tend to use a more visuospatial strategy to encode memory. In addition, novel analysis methods of brain networks can provide useful information about the gender specifics of brain functions. Gender‐related pseudo‐color maps constructed from all channels of average HbO₂ activity during low‐ and high‐load tasks (from 0 to 6 seconds after beginning).