Monochromatic subdiffusive spatial frequency domain imaging provides in‐situ sensitivity to intratumoral morphological heterogeneity in a murine model

For the first time, spatially resolved quantitative metrics of light scattering recovered with sub‐diffusive spatial frequency domain imaging (sd‐SFDI) are shown to be sensitive to changes in intratumoral morphology and viability by direct comparison to histopathological analysis. Two freshly excised subcutaneous murine tumor cross‐sections were measured with sd‐SFDI, and recovered optical scatter parameter maps were co‐registered to whole mount histology. Unique clustering of the optical scatter parameters vs. γ (i.e. diffuse scattering vs. relative backscattering) evaluated at a single wavelength showed complete separation between regions of viable tumor, aggresive tumor with stromal growth, varying levels of necrotic tumor, and also peritumor muscle. The results suggest that with further technical development, sd‐SFDI may represent a non‐destructive screening tool for analysis of excised tissue or a non‐invasive approach to investigate suspicious lesions without the need for exogenous labels or spectrally resolved imaging.

     

Near‐infrared human finger measurements based on self‐calibration point: Simulation and in vivo experiments

Near‐infrared light allows measuring tissue oxygenation. These measurements relay on oxygenation‐dependent absorption spectral changes. However, the tissue scattering, which is also spectral dependent, introduces an intrinsic error. Most methods focus on the volume reflectance from a semi‐infinite sample. We have proposed examining the full scattering profile (FSP), which is the angular intensity distribution. A point was found, that is, the iso‐path length (IPL) point, which is not dependent on the tissue scattering, and can serve for self‐calibration. This point is geometric dependent, hence in cylindrical tissues depends solely on the diameter. In this work, we examine an elliptic tissue cross section via Monte Carlo simulation. We have found that the IPL point of an elliptic tissue cross section is indifferent to the input illumination orientation. Furthermore, the IPL point is the same as in a circular cross section with a radius equal to the effective ellipse radius. This is despite the fact that the FSPs of the circular and elliptical cross sections are different. Hence, changing the orientation of the input illumination reveals the IPL point. In order to demonstrate this experimentally, the FSPs of a few female fingers were measured at 2 perpendicular orientations. The crossing point between these FSPs was found equivalent to the IPL point of a cylindrical phantom with a radius similar to the effective radius. The findings of this work will allow accurate pulse oximetry assessment of blood saturation.      

An improved algorithm for fast resonant Mie scatter correction of infrared spectra of cells and tissues

Mie scattering effects create serious problems for the interpretation of Fourier‐transform infrared spectroscopy spectra of single cells and tissues. During recent years, different techniques were proposed to retrieve pure absorbance spectra from spectra with Mie distortions. Recently, we published an iterative algorithm for correcting Mie scattering in spectra of single cells and tissues, which we called “the fast resonant Mie scatter correction algorithm.” The algorithm is based on extended multiplicative signal correction (EMSC) and employs a meta‐model for a parameter range of refractive index and size parameters. In the present study, we suggest several improvements of the algorithm. We demonstrate that the improved algorithm reestablishes chemical features of the measured spectra, and show that it tends away from the reference spectrum employed in the EMSC. We suggest strategies for choosing parameter ranges and other model parameters such as the number of principal components of the meta‐model and the number of iterations. We demonstrate that the suggested algorithm optimizes an error function of the refractive index in a forward Mie model. We suggest a stop criterion for the iterative algorithm based on the error function of the forward model.      

Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

Fingerprint‐to‐CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope

Stimulated Raman scattering (SRS) microscopy is a label‐free method generating images based on chemical contrast within samples, and has already shown its great potential for high‐sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio‐samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub‐cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto‐optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon‐Hydrogen (CH)‐stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm^?1, is demonstrated.     

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.     

Quantitative discrimination of NPC cell lines using optical coherence tomography

We tried to explore the intrinsic differences in the optical properties of the four representative NPC cell lines on the models of radiobiology and metastasis by OCT. The scattering coefficients and anisotropies were extracted by fitting the average a‐scan attenuation curves based on the multiple scatter effect. The values of scattering coefficients and anisotropy factors were 5.21 ± 0.11, 5.30 ± 0.09, 5.92 ± 0.21, 6.97 ± 0.22, and 0.892 ± 0.009, 0.886 ± 0.006, 0.884 ± 0.009, 0.86 ± 0.01 for CNE1, CNE2, 5‐8F and 6‐10B pellets (p < 0.05, P = 0.07 for CNE1 and CNE2), respectively. The results showed that the radiobiology and metastasis cell's model could be distinguished obviously; which implied that the corresponding types of NPC tissue might be potentially differentiated by OCT. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Skin and subcutaneous fat morphology alterations under the LED or laser treatment in rats in vivo

The main objective of this work is to quantify the impact of photodynamic/photothermal treatment by using visible LED and NIR laser irradiation through the skin of subcutaneous fat in vivo followed up by tissue sampling and histology. The optical method may provide reduction of regional or site‐specific accumulations of abdominal or subcutaneous adipose tissue precisely and least‐invasively by inducing cell apoptosis and controlled necrosis of fat tissue. As photodynamic/photothermal adipose tissue sensitizers Brilliant Green (BG) or Indocyanine Green (ICG) dyes were injected subcutaneously in rats. The CW LED device (625 nm) or CW diode laser (808 nm) were used as light sources, respectively. Biopsies of skin together with subcutaneous tissues were taken for histology. The combined action BG‐staining and LED‐irradiation (BG + LED) or ICG‐staining and NIR‐laser irradiation (ICG + NIR) causes pronounced signs of damage of adipose tissue characterized by a strong stretching, thinning, folding and undulating of cell membranes and appearance of necrotic areas. As a posttreatment after 14 days only connective tissue was observed at the site of necrotic areas. The data obtained are important for safe light treatment of site‐specific fat accumulations, including cellulite. This work provides a basis for the development of fat lipolysis technologies and to move them to clinical applications. Schematics of animal experiment.      

Generalized spectral light scatter models of diverse bacterial colony morphologies

An optical forward‐scatter model was generalized to encompass the diverse nature of bacterial colony morphologies and the spectral information. According to the model, the colony shape and the wavelength of incident light significantly affect the characteristics of a forward elastic‐light‐scattering pattern. To study the relationship between the colony morphology and the scattering pattern, three‐dimensional colony models were generated in various morphologies. The propagation of light passing through the colony model was then simulated. In validation of the theoretical modeling, the scattering patterns of three bacterial genera, Staphylococcus, Exiguobacterium and Bacillus, which grow into colonies having convex, crateriform and flat elevations, respectively, were qualitatively compared to the simulated scattering patterns. The strong correlations observed between simulated and experimental patterns validated the scatter model. In addition, spectral effect on the scattering pattern was studied using the scatter model, and experimentally investigated using Staphylococcus, whose colony has circular form and convex elevation. Both simulation and experiment showed that changes in wavelength affected the overall pattern size and the number of rings.     

fMRI Validation of fNIRS Measurements During a Naturalistic Task

We present a method to compare brain activity recorded with near-infrared spectroscopy (fNIRS) in a dance video game task to that recorded in a reduced version of the task using fMRI (functional magnetic resonance imaging). Recently, it has been shown that fNIRS can accurately record functional brain activities equivalent to those concurrently recorded with functional magnetic resonance imaging for classic psychophysical tasks and simple finger tapping paradigms. However, an often quoted benefit of fNIRS is that the technique allows for studying neural mechanisms of complex, naturalistic behaviors that are not possible using the constrained environment of fMRI. Our goal was to extend the findings of previous studies that have shown high correlation between concurrently recorded fNIRS and fMRI signals to compare neural recordings obtained in fMRI procedures to those separately obtained in naturalistic fNIRS experiments. Specifically, we developed a modified version of the dance video game Dance Dance Revolution (DDR) to be compatible with both fMRI and fNIRS imaging procedures. In this methodology we explain the modifications to the software and hardware for compatibility with each technique as well as the scanning and calibration procedures used to obtain representative results. The results of the study show a task-related increase in oxyhemoglobin in both modalities and demonstrate that it is possible to replicate the findings of fMRI using fNIRS in a naturalistic task. This technique represents a methodology to compare fMRI imaging paradigms which utilize a reduced-world environment to fNIRS in closer approximation to naturalistic, full-body activities and behaviors. Further development of this technique may apply to neurodegenerative diseases, such as Parkinson’s disease, late states of dementia, or those with magnetic susceptibility which are contraindicated for fMRI scanning.     

Simulation‐selection‐extrapolation: Estimation in high‐dimensional errors‐in‐variables models

Errors‐in‐variables models in high‐dimensional settings pose two challenges in application. First, the number of observed covariates is larger than the sample size, while only a small number of covariates are true predictors under an assumption of model sparsity. Second, the presence of measurement error can result in severely biased parameter estimates, and also affects the ability of penalized methods such as the lasso to recover the true sparsity pattern. A new estimation procedure called SIMulation‐SELection‐EXtrapolation (SIMSELEX) is proposed. This procedure makes double use of lasso methodology. First, the lasso is used to estimate sparse solutions in the simulation step, after which a group lasso is implemented to do variable selection. The SIMSELEX estimator is shown to perform well in variable selection, and has significantly lower estimation error than naive estimators that ignore measurement error. SIMSELEX can be applied in a variety of errors‐in‐variables settings, including linear models, generalized linear models, and Cox survival models. It is furthermore shown in the Supporting Information how SIMSELEX can be applied to spline‐based regression models. A simulation study is conducted to compare the SIMSELEX estimators to existing methods in the linear and logistic model settings, and to evaluate performance compared to naive methods in the Cox and spline models. Finally, the method is used to analyze a microarray dataset that contains gene expression measurements of favorable histology Wilms tumors.     

Controlling the near‐infrared transparency of costal cartilage by impregnation with clearing agents and magnetite nanoparticles

Penetration depth of near‐infrared laser radiation to costal cartilage is controlled by the tissue absorption and scattering, and it is the critical parameter to provide the relaxation of mechanical stress throughout the whole thickness of cartilage implant. To enhance the penetration for the laser radiation on 1.56 μm, the optical clearing solutions of glycerol and fructose of various concentrations are tested. The effective and reversible tissue clearance was achieved. However, the increasing absorption of radiation should be concerned: 5°C‐8°C increase of tissue temperature was detected. Laser parameters used for stress relaxation in cartilage should be optimized when applying optical clearing agents. To concentrate the absorption in the superficial tissue layers, magnetite nanoparticle (NP) dispersions with the mean size 95 ± 5 nm and concentration 3.9 ± 1.1 × 10¹¹ particles/mL are applied. The significant increase in the tissue heating rate was observed along with the decrease in its transparency. Using NPs the respective laser power can be decreased, allowing us to obtain the working temperature locally with reduced thermal effect on the surrounding tissue.      

Dose‐dependent study of effects of 532‐nm continuous wave laser on rat skin: A mechanistic insight

Visible lasers emitting in the green spectral region are being routinely employed in various medical and defense fields namely treatment of pigmented lesions, tattoo inks, port wine stains, dazzling the target or mob dispersal. Despite their increasing applications, lasers also tend to pose occupational hazards to operators, ancillary personnel, individuals undergoing laser therapies. This study was aimed at investigating the effects of different doses of 532‐nm continuous wave laser on rat skin. The present study demonstrated that higher fluences of 532‐nm continuous wave (CW) laser induces significant tissue damage through induction of tumor necrosis factor‐α, cyclooxygenase‐2, tumor protein (p53), PARP 1, caspase3 which in turn leads to tissue damage and cell death. Furthermore, level of heat shock proteins, pAkt were found up‐regulated as a cope up response to laser‐induced stress. On the basis of the findings, irradiation with 532‐nm CW laser up to 2.5 J/cm² was found within the safe exposure limits. Thus, it is probably the first attempt to demonstrate the tissue damage induced by 532‐nm CW laser on skin, which may help in choosing safe laser dose for certain skin‐based applications and evolving methods to ameliorate laser‐inflicted injuries.     

Split‐root systems applied to the study of the legume‐rhizobial symbiosis: What have we learned?

Split‐root system (SRS) approaches allow the differential treatment of separate and independent root systems, while sharing a common aerial part. As such, SRS is a useful tool for the discrimination of systemic (shoot origin) versus local (root/nodule origin) regulation mechanisms. This type of approach is particularly useful when studying the complex regulatory mechanisms governing the symbiosis established between legumes and Rhizobium bacteria. The current work provides an overview of the main insights gained from the application of SRS approaches to understand how nodule number (nodulation autoregulation) and nitrogen fixation are controlled both under non‐stressful conditions and in response to a variety of stresses. Nodule number appears to be mainly controlled at the systemic level through a signal which is produced by nodule/root tissue, translocated to the shoot, and transmitted back to the root system, involving shoot Leu‐rich repeat receptor‐like kinases. In contrast, both local and systemic mechanisms have been shown to operate for the regulation of nitrogenase activity in nodules. Under drought and heavy metal stress, the regulation is mostly local, whereas the application of exogenous nitrogen seems to exert a regulation of nitrogen fixation both at the local and systemic levels.     

K‐distribution three‐dimensional mapping of biological tissues in optical coherence tomography

Probability density function (PDF) analysis with K‐distribution model of optical coherence tomography (OCT) intensity signals has previously yielded a good representation of the average number of scatterers in a coherence volume for microspheres‐in‐water systems, and has shown initial promise for biological tissue characterization. In this work, we extend these previous findings, based on single point M‐mode or two‐dimenstional slice analysis, to full three‐dimensional (3D) imaging maps of the shape parameter α of the K‐distribution PDF. After selecting a suitably sized 3D evaluation window, and verifying methodology in phantoms, the resultant parametric α images obtained in different animal tissues (rat liver and brain) show new contrasting ability not seen in conventional OCT intensity images.      

Skeletal muscle fat oxidation is increased in African-American and white women after 10 days of endurance exercise training

Objective: Obesity is associated with lower rates of skeletal muscle fatty acid oxidation (FAO), which is linked to insulin resistance. FAO is reduced further in obese African‐American (AAW) vs. white women (CW) and may also be lower in lean AAW vs. CW. In lean CW, endurance exercise training (EET) elevates the oxidative capacity of skeletal muscle. Therefore, we determined whether EET would elevate skeletal muscle FAO similarly in AAW and CW with a lower lipid oxidative capacity. Research Methods and Procedures: In vitro rates of FAO were assessed in rectus abdominus muscle strips using [1‐¹⁴C] palmitate (Pal) from lean AAW [BMI = 24.2 ± 0.9 (standard error) kg/m²] and CW (23.6 ± 0.8 kg/m²) undergoing voluntary abdominal surgery. Lean AAW (22 ± 0.9 kg/m²) and CW (24 ± 0.8 kg/m²) and obese AAW (36 ± 1.2 kg/m²) and CW (40 ± 1.3 kg/m²) underwent 10 consecutive days of EET on a cycle ergometer (60 min/d, 75% peak oxygen uptake). FAO was measured in vastus lateralis homogenates as captured ¹⁴CO₂ using [1‐¹⁴C] Pal, palmitoyl‐CoA (Pal‐CoA), and palmityl‐carnitine (Pal‐Car). Results: Muscle strip experiments showed suppressed rates of FAO (p = 0.03) in lean AAW vs. CW. EET increased the rates of skeletal muscle Pal oxidation (p = 0.05) in both lean AAW and CW. In obese subjects, Pre‐EET Pal (but not Pal‐CoA or Pal‐Car) oxidation was lower (p = 0.05) in AAW vs. CW. EET increased Pal oxidation 100% in obese AAW (p < 0.05) and 59% (p < 0.05) in obese CW. Similar increases (p < 0.05) in post‐EET FAO were observed for Pal‐CoA and Pal‐Car in both groups. Discussion: Both lean and obese AAW possess a lower capacity for skeletal muscle FAO, but EET increases FAO similarly in both AAW and CW. These data suggest the use of EET for treatment against obesity and diabetes for both AAW and CW.     

Chemical imaging of fresh vascular smooth muscle cell response by epi‐detected stimulated Raman scattering

An understanding of deformation of cardiovascular tissue under hemodynamic load is crucial for understanding the health and disease of blood vessels. In the present work, an epi‐detected stimulated Raman scattering (epi‐SRS) imaging platform was designed for in situ functional imaging of vascular smooth muscle cells (VMSCs) in fresh coronary arteries. For the first time, the pressure‐induced morphological deformation of fresh VSMCs was imaged with no fixation and in a label‐free manner. The relation between the loading pressure and the morphological parameters, including angle and length of the VSMCs, were apparent. The morphological responses of VMSCs to drug treatment were also explored, to demonstrate the capability of functional imaging for VSMCs by this method. The time‐course imaging revealed the drug induced change in angle and length of VSMCs. The present study provides a better understanding of the biomechanical framework of blood vessels, as well as their responses to external stimulations, which are fundamental for developing new strategies for cardiovascular disease treatment.      

Light distribution and thermal effects in the rat brain under optogenetic stimulation

Optical brain stimulation gained a lot of attention in neuroscience due to its superior cell‐type specificity. In the design of illumination strategies, predicting the light propagation in a specific tissue is essential and requires knowledge of the optical properties of that tissue. We present the estimated absorption and reduced scattering in rodent brain tissue using non‐destructive contact spatially resolved spectroscopy (cSRS). The obtained absorption and scattering in the cortex, hippocampus and striatum are similar, but lower than in the thalamus, leading to a less deep but broader light penetration profile in the thalamus. Next, the light distribution was investigated for different stimulation protocols relevant for fiber‐optic based optogenetic experiments, using Monte Carlo simulation. A protocol specific analysis is proposed to evaluate the potential of thermally induced side effects.

     

1064 nm acoustic resolution photoacoustic microscopy

Photoacoustic imaging is a noninvasive imaging technique having the advantages of high‐optical contrast and good acoustic resolution at improved imaging depths. Light transport in biological tissues is mainly characterized by strong optical scattering and absorption. Photoacoustic microscopy is capable of achieving high‐resolution images at greater depth compared to conventional optical microscopy methods. In this work, we have developed a high‐resolution, acoustic resolution photoacoustic microscopy (AR‐PAM) system in the near infra‐red (NIR) window II (NIR‐II, eg, 1064 nm) for deep tissue imaging. Higher imaging depth is achieved as the tissue scattering at 1064 nm is lesser compared to visible or near infrared window‐I (NIR‐I). Our developed system can provide a lateral resolution of 130 μm, axial resolution of 57 μm, and image up to 11 mm deep in biological tissues. This 1064‐AR‐PAM system was used for imaging sentinel lymph node and the lymph vessel in rat. Urinary bladder of rat filled with black ink was also imaged to validate the feasibility of the developed system to study deeply seated organs.      

Label‐free stimulated Raman scattering imaging reveals silicone breast implant material in tissue

Millions of women worldwide have silicone breast implants. It has been reported that implant failure occurs in approximately a tenth of patients within 10 years, and the consequences of dissemination of silicone debris are poorly understood. Currently, silicone detection in histopathological slides is based on morphological features as no specific immunohistochemical technique is available. Here, we show the feasibility and sensitivity of stimulated Raman scattering (SRS) imaging to specifically detect silicone material in stained histopathological slides, without additional sample treatment. Histology slides of four periprosthetic capsules from different implant types were obtained after explantation, as well as an enlarged axillary lymph node from a patient with a ruptured implant. SRS images coregistered with bright‐field images revealed the distribution and quantity of silicone material in the tissue. Fast and high‐resolution imaging of histology slides with molecular specificity using SRS provides an opportunity to investigate the role of silicone debris in the pathophysiology of implant‐linked diseases.