Optimized skin optical clearing for optical coherence tomography monitoring of encapsulated drug delivery through the hair follicles

Hair follicles (HF) represent a drug delivery reservoir for improved treatment of skin disorders. Although various particulate systems play an important role in HF‐targeting, their optical monitoring in skin is challenging due to strong light scattering. Optical clearing is an effective approach allowing the increasing of particle detection depth in skin. The enhancement of optical probing depth (OPD) and optical detection depth (ODD) of particle localization using optical coherence tomography (OCT) was evaluated under application of various optical clearing agents (OCAs) together with skin permeability enhancers ex vivo in rats. Efficient OPD increasing was demonstrated for all investigated OCAs. However, skin dehydration under action of hyperosmotic agents led to the worsening of OCT‐contrast in dermis decreasing the ODD. Lipophilic agents provided optical clearing of epidermis without its dehydration. The highest ODD was obtained at application of a PEG‐400/oleic acid mixture. This OCA was tested in vivo showing beneficial ODD and OPD enhancement.     

Visible‐near infrared‐II skull optical clearing window for in vivo cortical vasculature imaging and targeted manipulation

Skull optical clearing window permits us to perform in vivo cortical imaging without craniotomy, but mainly limits to visible (vis)‐near infrared (NIR)‐I light imaging. If the skull optical clearing window is available for NIR‐II, the imaging depth will be further enhanced. Herein, we developed a vis‐NIR‐II skull optical clearing agents with deuterium oxide instead of water, which could make the skull transparent in the range of visible to NIR‐II. Using a NIR‐II excited third harmonic generation microscope, the cortical vasculature of mice could be clearly distinguished even at the depth of 650 μm through the vis‐NIR‐II skull clearing window. The imaging depth after clearing is close to that without skull, and increases by three times through turbid skull. Furthermore, the new skull optical clearing window promises to realize NIR‐II laser‐induced targeted injury of cortical single vessel. This work enhances the ability of NIR‐II excited nonlinear imaging techniques for accessing to cortical neurovasculature in deep tissue.     

Hydrogen bound water profiles in the skin influenced by optical clearing molecular agents—Quantitative analysis using confocal Raman microscopy

Confocal Raman microscopy has been used to measure depth‐dependent profiles of porcine skin ex vivo in the high wavenumber region after application of molecular optical clearing agents (OCAs). Glycerol (70%) and iohexol (100% Omnipaque [300]) water solutions were used as OCAs and topically applied to porcine ear skin for 30 and 60 minutes. Using Gaussian function–based deconvolution, the changes of hydrogen bound water molecule types have been microscopically analyzed down to the depth of 200 μm. Results show that both OCAs induced skin dehydration (reduction of total water), which is 51.3% for glycerol (60 minutes), 33.1% for glycerol (30 minutes), 8.3% for Omnipaque (60 minutes) and 4.4% for Omnipaque (30 minutes), on average for the 40 to 200 μm depths. Among the water types in the skin, the following reduction was observed in concentration of weakly bound (51.1%, 33.2%, 7.5% and 4.6%), strongly bound (50.4%, 33.0%, 7.9% and 3.4%), tightly bound (63.6%, 42.3%, 26.1% and 12.9%) and unbound (55.4%, 28.7%, 10.1% and 5.9%) water types on average for the 40 to 200 μm depths, post application of glycerol (60 minutes), glycerol (30 minutes), Omnipaque (60 minutes) and Omnipaque (30 minutes), respectively. As most concentrated in the skin, weakly and strongly bound water types are preferentially involved in the OCA‐induced water flux in the skin, and thus, are responsible for optical clearing efficiency.      

Dynamic change in optical properties of a nanoparticle embedded tumor phantom for plasmonic photothermal cancer therapeutics

In this study, the temporal dynamic changes in optical properties of gold nanorods (GNR) embedded tumor phantom, during photothermal interaction, are reported for plasmonic photothermal therapeutics. Tumor mimicking bilayer phantoms were prepared by using 1% agarose incorporated with 0.1% coffee powder, 0.3% intralipid solution as epidermis layer; 3% intralipid solution and 0.3% human hemoglobin (Hb) powder as dermis layer. On incorporating GNRs of concentrations 10, 20, and 40 μg/ml within the phantom, the absorption coefficients increases 4–8 times, while there is minimal change in the reduced scattering coefficients. Further the absorption coefficient increased by ~8% with the incorporation of GNRs of concentration 40 μg/ml, while no considerable dynamic change in the optical properties is observed for the phantom embedded with GNRs of concentrations 10, and 20 μg/ml. The discussed results are useful for the selection of GNRs dose for pre-treatment planning of plasmonic photothermal cancer therapeutics.     

Reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing

Wavefront shaping can compensate the wavefront distortions in deep tissue focusing, leading to an improved penetration depth. However, when using the backscattered signals as the feedback, unexpected compensation bias may be introduced, resulting in focusing position deviations or even no focus in the illumination focal plane. Here we investigated the reliability of wavefront shaping based on coherent optical adaptive technique in deep tissue focusing by measuring the position deviations between the foci in the illumination focal plane and the epi‐detection plane. The experimental results show that when the penetration depth reaches 150 μm in mouse brain tissue (with scattering coefficient ~22.42 mm^?1) using a 488 nm laser and an objective lens with 0.75 numerical aperture, the center of the real focus will deviate out of one radius range of the Airy disk while the optimized focus in the epi‐detection plane maintained basically at the center. With the penetration depth increases, the peak to background ratio of the focus in the illumination focal plane decreases faster than that in the epi‐detection plane. The results indicate that when the penetration depth reaches 150 μm, feedback based on backscattered signals will make wavefront shaping lose its reliability, which may provide a guidance for applications of non‐invasive precise optogenetics or deep tissue optical stimulation using wavefront shaping methods. A, Intensity distribution in the epi‐detection plane and the illumination focal plane before and after correction, corresponding to brain sections with 250 and 300 μm thickness, respectively. Scale bar is 2 μm. B, Averaged focusing deviations in the epi‐detection plane (optimized) and the illumination focal plane (monitored) after compensation. The unit of the ordinate is one Airy disk diameter. Black dashed line represents one Airy disk radius. Bars represent the SE of each measurement set.      

Acoustic resolution photoacoustic microscopy based on microelectromechanical systems scanner

Photoacoustic microscopy (PAM) can be classified as optical resolution (OR)‐PAM and acoustic resolution (AR)‐PAM depending on the type of resolution achieved. Using microelectromechanical systems (MEMS) scanner, high‐speed OR‐PAM system was developed earlier. Depth of imaging limits the use of OR‐PAM technology for many preclinical and clinical imaging applications. Here, we demonstrate the use of a high‐speed MEMS scanner for AR‐PAM imaging. Lateral resolution of 84 μm and an axial resolution of 27 μm with ~2.7 mm imaging depth was achieved using a 50 MHz transducer‐based AR‐PAM system. Use of a higher frequency transducer at 75 MHz has further improved the resolution characteristics of the system with a reduction in imaging depth and a lateral resolution of 53 μm and an axial resolution of 18 μm with ~1.8 mm imaging depth was achieved. Using the two‐axis MEMS scanner a 2 × 2 .5 mm² area was imaged in 3 seconds. The capability of achieving acoustic resolution images using the MEMS scanner makes it beneficial for the development of high‐speed miniaturized systems for deeper tissue imaging.      

Tissue clearing‐based method for unobstructed three‐dimensional imaging of mouse penis with subcellular resolution

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous‐ (clear, unobstructed brain imaging cocktails and computational analysis) and solvent‐based (fluorescence‐preserving capability imaging of solvent‐cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three‐dimensional structure of murine penis fibrocartilage.     

Impact of optical clearing on ex vivo human skin optical properties characterized by spatially resolved multimodal spectroscopy

A spatially resolved multimodal spectroscopic device was used on a two-layered “hybrid” model made of ex vivo skin and fluorescent gel to investigate the effect of skin optical clearing on the depth sensitivity of optical spectroscopy. Time kinetics of fluorescence and diffuse reflectance spectra were acquired in four experimental conditions: with optical clearing agent (OCA) 1 made of polyethylene glycol 400 (PEG-400), propylene glycol and sucrose; with OCA 2 made of PEG-400 and dimethyl sulfoxide (DMSO); with saline solution as control and a “dry” condition. An increase in the gel fluorescence back reflected intensity was measured after optical clearing. Effect of OCA 2 turned out to be stronger than that of OCA 1, possibly due to DMSO impact on the stratum corneum keratin conformation. Complementary experimental results showed increased light transmittance through the skin and confirmed that the improvement in the depth sensitivity of the multimodal spectroscopic approach is related not only to the dehydration and refractive indices matching due to optical clearing, but also to the mechanical compression of tissues caused by the application of the spectroscopic probe.     

Miniaturized all fiber probe for optical coherence tomography and pH detection of biological tissue

We present a novel all-fiber probe with 710-μm outside diameter for combined optical coherence tomography and pH detection. In cancer surgery, a significant challenge is how to completely remove the malignant tumor without cutting too much normal tissue. The difference between cancer tissue and normal tissue not only lies in morphology and structure but also in tissue pH, where malignant tissue has a lower pH. This dual-modality probe combined optical coherence tomography and pH detection of biological tissue, is expected to determine whether the tissue is cancerous quickly and accurately. The probe utilizes a typical three-segment structure (double-clad fiber - no-core fiber - graded-index fiber). We obtained a lateral resolution of ~10.6 μm, a working distance of ~506 μm and a pH measurement accuracy of 0.01 pH unit for the probe. The performance of the all-fiber probe was verified through an ex vivo experiment using the porcine brain specimen.     

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.      

Optogenetic approaches for termination of ventricular tachyarrhythmias after myocardial infarction in rats in vivo

Cardiac optogenetics facilitates the painless manipulation of the heart with optical energy and was recently shown to terminate ventricular tachycardia (VT) in explanted mice heart. This study aimed to evaluate the optogenetic‐based termination of induced VT under ischemia in an open‐chest rat model and to develop an optimal, optical‐manipulation procedure. VT was induced by burst stimulation after ligation of the left anterior descending coronary artery, and the termination effects of the optical manipulation, including electrical anti‐tachycardia pacing (ATP) and spontaneous recovery, were tested. Among different multisegment optical modes, four repeated illuminations of 1000 ms in duration with 1‐second interval at a 20‐times intensity threshold on the right ventricle achieved the highest termination rate of 86.14% ± 4.145%, higher than that achieved by ATP and spontaneous termination. We demonstrated that optogenetic‐based cardioversion is feasible and effective in vivo, with the underlying mechanism involving the light‐triggered, ChR2‐induced depolarization of the illuminated myocardium, in turn generating an excitation that disrupts the preexisting reentrant wave front.     

Broadband spectral verification of optical clearing reversibility in lung tissue

The increase of tissue transparency through sequential optical immersion clearing treatments and treatment reversibility have high interest for clinical applications. To evaluate the clearing reversibility in a broad spectral range and the magnitude of the transparency created by a second treatment, the present study consisted on measuring the spectral collimated transmittance of lung tissues during a sequence of two treatments with electronic cigarette (e-cig) fluid, which was intercalated with an immersion in saline. The saline immersion clearly reverted the clearing effect in the lung tissue in the spectral range between 220 and 1000 nm. By a later application of a second treatment with the e-cig fluid, the magnitude of the optical clearing effect was observed to be about the double as the one observed in the first treatment, showing that the molecules of the optical clearing agent might have converted some bound water into mobile water during the first treatment.     

Stimulation of the active transport of serotonin into mouse platelets by the sulfhydryl oxidizing agent diamide

The purpose of this study was to determine the effects of diamide, a reversible sulfhydryl oxidizing agent, on the transport of serotonin (5-HT) by mouse platelets. Diamide produced a concentration-dependent (10–200 μM) stimulation of 5-HT transport that was rapid and sustained over 0–10 minutes of incubation. When platelets were incubated with diamide (10–200 μM) in the presence of glucose, the content of reduced glutathione was significantly decreased only at a final concentration of 200 μM, while washed platelets incubated with diamide (10–200 μM), in the absence of glucose, had a significant concentration-dependent decrease in their content of reduced glutathione. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked diamide-induced stimulation of 5-HT transport. The kinetics of 5-HT transport showed that diamide caused a marked increase in the maximal rate of transport (V_max control = 28.4 ± 1.4 vs. V_max diamide = 60.9 ± 4.1 pM/10⁸ platelets/4 min) but did not significantly alter the K_m values. Ouabain, an inhibitor of platelet Na⁺-K⁺ ATPase, blocked the stimulation by diamide in a concentration-dependent manner. Dithiothreitol, a disulfide reducing agent, was able to partially reverse the stimulation of platelet 5-HT transport caused by diamide. This study has shown that diamide can stimulate the active transport of 5-HT by mouse platelets and suggests a possible role for free sulfhydryl groups in the regulation of this process.     

Sizing the pore of the volume-sensitive anion channel by differential polymer partitioning

Partitioning of ethylene glycol and its polymeric forms into the pore of the volume-sensitive outwardly rectifying (VSOR) anion channel was studied to assess the pore size. Polyethylene glycol (PEG) PEG 200-300 (Rh = 0.27-0.53 nm) effectively suppressed the single-channel currents, whereas PEG 400-4000 (Rh = 0.62-1.91 nm) had little or no effect. Since all the molecules tested effectively decreased electric conductivity of the bulk solution, the observed differential effects between PEG 200-300 and PEG 400-4000 on the VSOR single-channel current are due to their limited partitioning into the channel lumen. The cut-off radius of the VSOR channel pore was assessed to be 0.63 nm.     

Volume Changes of Individual Melanosomes Measured by Scanning Force Microscopy

Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G‐protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three‐dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P<0.05, n=200). The average ellipsoidal volume was 0.14±0.01 (aggregated) and 0.17±0.01 μm³ (dispersed), a difference of 18%. The average major diameter was 810±20 and 880±20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non‐destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.     

Quantification of perfusion and metabolism in an autism mouse model assessed by diffuse correlation spectroscopy and near-infrared spectroscopy

There is a need for quantitative biomarkers for early diagnosis of autism. Cerebral blood flow and oxidative metabolism parameters may show superior contrasts for improved characterization. Diffuse correlation spectroscopy (DCS) has been shown to be reliable method to obtain cerebral blood flow contrast in animals and humans. Thus, in this study, we evaluated the combination of DCS and fNIRS in an established autism mouse model. Our results indicate that autistic group had significantly (P = .001) lower (~40%) blood flow (1.16 ± 0.26) × 10⁻⁸ cm²/s), and significantly (P = .015) lower (~70%) oxidative metabolism (52.4 ± 16.6 μmol/100 g/min) compared to control group ([1.93 ± 0.74] × 10⁻⁸ cm²/s, 177.2 ± 45.8 μmol/100 g/min, respectively). These results suggest that the combination of DCS and fNIRS can provide hemodynamic and metabolic contrasts for in vivo assessment of autism pathological conditions noninvasively.     

Is oblique scanning laser ophthalmoscope applicable to human ocular optics? A feasibility study using an eye model for volumetric imaging

Oblique scanning laser ophthalmoscopy (oSLO) is a novel imaging modality to provide volumetric retinal imaging without depth sectioning over a large field of view (FOV). It has been successfully demonstrated in vivo in rodent eyes for volumetric fluorescein angiography (vFA). However, engineering oSLO for human retinal imaging is challenging because of the low numerical aperture (NA) of human ocular optics. To overcome this challenge, we implement optical designs to (a) increase the angle of the intermediate image under Scheimpflug condition, and (b) expand the magnification in the depth dimension with cylindrical lens to enable sufficient sampling density. In addition, we adopt a scanning‐and‐descaning strategy, resulting in a compact oSLO system. We experimentally show that the current setup can achieve a FOV of ~3 × 6 × 0.8 mm³, and the transverse and axial resolutions of 7 and 41 μm, respectively. This feasibility study serves an important step for future in vivo human retinal imaging.     

Characterization of optical clearing mechanisms in muscle during treatment with glycerol and gadobutrol solutions

The recent increasing interest in the application of radiology contrasting agents to create transparency in biological tissues implies that the diffusion properties of those agents need evaluation. The comparison of those properties with the ones obtained for other optical clearing agents allows to perform an optimized agent selection to create optimized transparency in clinical applications. In this study, the evaluation and comparison of the diffusion properties of gadobutrol and glycerol in skeletal muscle was made, showing that although gadobutrol has a higher molar mass than glycerol, its low viscosity allows for a faster diffusion in the muscle. The characterization of the tissue dehydration and refractive index matching mechanisms of optical clearing was made in skeletal muscle, namely by the estimation of the diffusion coefficients for water, glycerol and gadobutrol. The estimated tortuosity values of glycerol (2.2) and of gadobutrol (1.7) showed a longer path-length for glycerol in the muscle.     

The use of intensity‐based Doppler variance method for single vessel response to functional neurovascular activation

This study presents 1 use of optical coherence tomography (OCT) angiography technique to examine neurovascular coupling effect. Repeated B‐scans OCT recording is performed on the rat somatosensory cortex with cranial window preparation while its contralateral forepaw is electrically stimulated to activate the neurons in rest. We use an intensity‐based Doppler variance (IBDV) algorithm mapped cerebral blood vessels in the cortex, and the temporal alteration in blood perfusion during neurovascular activation is analyzed using the proposed IBDV quantitative parameters. By using principal component analysis‐based Fuzzy C Means clustering method, the stimulus‐evoked vasomotion patterns were classified into 3 categories. We found that the response time of small vessels (resting diameter 14.9 ±6.6 μm), middle vessels (resting diameter 21.1 ±7.9 μm) and large vessels (resting diameter 50.7 ±6.5 μm) to achieve 5% change of vascular dilation after stimulation was 1.5, 2 and 5.5 seconds, respectively. Approximately 5% peak change of relative blood flow (RBF) in both small and middle vessels was observed. The large vessels react slowly and their responses nearly 4 seconds delayed, but no significant change in RBF of the large vessels was seen.      

Effects of Obesity on Substrate Utilization during Exercise

Objective: The capacity for lipid and carbohydrate (CHO) oxidation during exercise is important for energy partitioning and storage. This study examined the effects of obesity on lipid and CHO oxidation during exercise. Research Methods and Procedures: Seven obese and seven lean [body mass index (BMI), 33 ± 0.8 and 23.7 ± 1.2 kg/m², respectively] sedentary, middle‐aged men matched for aerobic capacity performed 60 minutes of cycle exercise at similar relative (50% Vo _2max) and absolute exercise intensities. Results: Obese men derived a greater proportion of their energy from fatty‐acid oxidation than lean men (43 ± 5% 31 ± 2%; p = 0.02). Plasma fatty‐acid oxidation determined from recovery of infused [0.15 μmol/kg fat‐free mass (FFM) per minute] [1‐¹³C]‐palmitate in breath CO₂ was similar for obese and lean men (8.4 ± 1.1 and 29 ± 15 μmol/kg FFM per minute). Nonplasma fatty‐acid oxidation, presumably, from intramuscular sources, was 50% higher in obese men than in lean men (10.0 ± 0.6 versus 6.6 ± 0.8 μmol/kg FFM per minute; p < 0.05). Systemic glucose disposal was similar in lean and obese groups (33 ± 8 and 29 ± 15 μmol/kg FFM per minute). However, the estimated rate of glycogen‐oxidation was 50% lower in obese than in lean men (61 ± 12 versus 90 ± 6 μmol/kg FFM per minute; p < 0.05). Discussion: During moderate exercise, obese sedentary men have increased rates of fatty‐acid oxidation from nonplasma sources and reduced rates of CHO oxidation, particularly muscle glycogen, compared with lean sedentary men.