In situ pulmonary mucus hydration assay using rotational and translational diffusion of gold nanorods with polarization-sensitive optical coherence tomography.

Significance: Assessing the nanostructure of polymer solutions and biofluids is broadly useful for understanding drug delivery and disease progression and for monitoring therapy. Aim: Our objective is to quantify bronchial mucus solids concentration (wt. %) during hypertonic saline (HTS) treatment in vitro via nanostructurally constrained diffusion of gold nanorods (GNRs) monitored by polarization-sensitive optical coherence tomography (PS-OCT). Approach: Using PS-OCT, we quantified GNR translational (DT) and rotational (DR) diffusion coefficients within polyethylene oxide solutions (0 to 3 wt. %) and human bronchial epithelial cell (hBEC) mucus (0 to 6.4 wt. %). Interpolation of DT and DR data is used to develop an assay to quantify mucus concentration. The assay is demonstrated on the mucus layer of an air-liquid interface hBEC culture during HTS treatment. Results: In polymer solutions and mucus, DT and DR monotonically decrease with increasing concentration. DR is more sensitive than DT to changes above 1.5 wt. % of mucus and exhibits less intrasample variability. Mucus on HTS-treated hBEC cultures exhibits dynamic mixing from cilia. A region of hard-packed mucus is revealed by DR measurements. Conclusions: The extended dynamic range afforded by simultaneous measurement of DT and DR of GNRs using PS-OCT enables resolving concentration of the bronchial mucus layer over a range from healthy to disease in depth and time during HTS treatment in vitro.

Diffusion tensor optical coherence tomography.

In situ measurements of diffusive particle transport provide insight into tissue architecture, drug delivery, and cellular function. Analogous to diffusion-tensor magnetic resonance imaging (DT-MRI), where the anisotropic diffusion of water molecules is mapped on the millimeter scale to elucidate the fibrous structure of tissue, here we propose diffusion-tensor optical coherence tomography (DT-OCT) for measuring directional diffusivity and flow of optically scattering particles within tissue. Because DT-OCT is sensitive to the sub-resolution motion of Brownian particles as they are constrained by tissue macromolecules, it has the potential to quantify nanoporous anisotropic tissue structure at micrometer resolution as relevant to extracellular matrices, neurons, and capillaries. Here we derive the principles of DT-OCT, relating the detected optical signal from a minimum of six probe beams with the six unique diffusion tensor and three flow vector components. The optimal geometry of the probe beams is determined given a finite numerical aperture, and a high-speed hardware implementation is proposed. Finally, Monte Carlo simulations are employed to assess the ability of the proposed DT-OCT system to quantify anisotropic diffusion of nanoparticles in a collagen matrix, an extracellular constituent that is known to become highly aligned during tumor development.

Diffusion-sensitive optical coherence tomography for real-time monitoring of mucus thinning treatments.

Mucus hydration (wt%) has become an increasingly useful metric in real-time assessment of respiratory health in diseases like cystic fibrosis and COPD, with higher wt% indicative of diseased states. However, available in vivo rheological techniques are lacking. Gold nanorods (GNRs) are attractive biological probes whose diffusion through tissue is sensitive to the correlation length of comprising biopolymers. Through employment of dynamic light scattering theory on OCT signals from GNRs, we find that weakly-constrained GNR diffusion predictably decreases with increasing wt% (more disease-like) mucus. Previously, we determined this method is robust against mucus transport on human bronchial epithelial (hBE) air-liquid interface cultures (R2=0.976). Here we introduce diffusion-sensitive OCT (DS-OCT), where we collect M-mode image ensembles, from which we derive depth- and temporally-resolved GNR diffusion rates. DS-OCT allows for real-time monitoring of changing GNR diffusion as a result of topically applied mucus-thinning agents, enabling monitoring of the dynamics of mucus hydration never before seen. Cultured human airway epithelial cells (Calu-3) with a layer of endogenous mucus were doped with topically deposited GNRs (80×22nm), and subsequently treated with hypertonic saline (HS) or isotonic saline (IS). DS-OCT provided imaging of the mucus thinning response up to a depth of 600µm with 4.65µm resolution, over a total of 8 minutes in increments of ≥3 seconds. For both IS and HS conditions, DS-OCT captured changes in the pattern of mucus hydration over time. DS-OCT opens a new window into understanding mechanisms of mucus thinning during treatment, enabling real-time efficacy feedback needed to optimize and tailor treatments for individual patients.

Magnetic and Plasmonic Contrast Agents in Optical Coherence Tomography.

Optical coherence tomography (OCT) has gained widespread application for many biomedical applications, yet the traditional array of contrast agents used in incoherent imaging modalities do not provide contrast in OCT. Owing to the high biocompatibility of iron oxides and noble metals, magnetic and plasmonic nanoparticles, respectively, have been developed as OCT contrast agents to enable a range of biological and pre-clinical studies. Here we provide a review of these developments within the past decade, including an overview of the physical contrast mechanisms and classes of OCT system hardware addons needed for magnetic and plasmonic nanoparticle contrast. A comparison of the wide variety of nanoparticle systems is also presented, where the figures of merit depend strongly upon the choice of biological application.

Imaging Extracellular Matrix Remodeling In Vitro by Diffusion-Sensitive Optical Coherence Tomography.

The mammary gland extracellular matrix (ECM) is comprised of biopolymers, primarily collagen I, that are created and maintained by stromal fibroblasts. ECM remodeling by fibroblasts results in changes in ECM fiber spacing (pores) that have been shown to play a critical role in the aggressiveness of breast cancer. However, minimally invasive methods to measure the spatial distribution of ECM pore areas within tissues and in vitro 3D culture models are currently lacking. We introduce diffusion-sensitive optical coherence tomography (DS-OCT) to image the nanoscale porosity of ECM by sensing weakly constrained diffusion of gold nanorods (GNRs). DS-OCT combines the principles of low-coherence interferometry and heterodyne dynamic light scattering. By collecting co- and cross-polarized light backscattered from GNRs within tissue culture, the ensemble-averaged translational self-diffusion rate, DT, of GNRs is resolved within ∼3 coherence volumes (10 × 5 µm, x × z). As GNRs are slowed by intermittent collisions with ECM fibers, DT is sensitive to ECM porosity on the size scale of their hydrodynamic diameter (∼46 nm). Here, we validate the utility of DS-OCT using pure collagen I gels and 3D mammary fibroblast cultures seeded in collagen/Matrigel, and associate differences in artificial ECM pore areas with gel concentration and cell seed density. Across all samples, DT was highly correlated with pore area obtained by scanning electron microscopy (R(2) = 0.968). We also demonstrate that DS-OCT can accurately map the spatial heterogeneity of layered samples. Importantly, DS-OCT of 3D mammary fibroblast cultures revealed the impact of fibroblast remodeling, where the spatial heterogeneity of matrix porosity was found to increase with cell density. This provides an unprecedented view into nanoscale changes in artificial ECM porosity over effective pore diameters ranging from ∼43 to 360 nm using a micron-scale optical imaging technique. In combination with the topical deposition of GNRs, the minimally invasive nature of DS-OCT makes this a promising technology for studying tissue remodeling processes.

Probing biological nanotopology via diffusion of weakly constrained plasmonic nanorods with optical coherence tomography.

Biological materials exhibit complex nanotopology, i.e., a composite liquid and solid phase structure that is heterogeneous on the nanoscale. The diffusion of nanoparticles in nanotopological environments can elucidate biophysical changes associated with pathogenesis and disease progression. However, there is a lack of methods that characterize nanoprobe diffusion and translate easily to in vivo studies. Here, we demonstrate a method based on optical coherence tomography (OCT) to depth-resolve diffusion of plasmon-resonant gold nanorods (GNRs) that are weakly constrained by the biological tissue. By using GNRs that are on the size scale of the polymeric mesh, their Brownian motion is minimally hindered by intermittent collisions with local macromolecules. OCT depth-resolves the particle-averaged translational diffusion coefficient (DT) of GNRs within each coherence volume, which is separable from the nonequilibrium motile activities of cells based on the unique polarized light-scattering properties of GNRs. We show how this enables minimally invasive imaging and monitoring of nanotopological changes in a variety of biological models, including extracellular matrix (ECM) remodeling as relevant to carcinogenesis, and dehydration of pulmonary mucus as relevant to cystic fibrosis. In 3D ECM models, DT of GNRs decreases with both increasing collagen concentration and cell density. Similarly, DT of GNRs is sensitive to human bronchial-epithelial mucus concentration over a physiologically relevant range. This novel method comprises a broad-based platform for studying heterogeneous nanotopology, as distinct from bulk viscoelasticity, in biological milieu.

Hollow steel tips for reducing distal fiber burn-back during thulium fiber laser lithotripsy.

The use of thulium fiber laser (TFL) as a potential alternative laser lithotripter to the clinical holmium:YAG laser is being studied. The TFL's Gaussian spatial beam profile provides efficient coupling of higher laser power into smaller core fibers without proximal fiber tip degradation. Smaller fiber diameters are more desirable, because they free up space in the single working channel of the ureteroscope for increased saline irrigation rates and allow maximum ureteroscope deflection. However, distal fiber tip degradation and "burn-back" increase as fiber diameter decreases due to both excessive temperatures and mechanical stress experienced during stone ablation. To eliminate fiber tip burn-back, the distal tip of a 150-µm core silica fiber was glued inside 1-cm-long steel tubing with fiber tip recessed 100, 250, 500, 1000, or 2000 µm inside the steel tubing to create the hollow-tip fiber. TFL pulse energy of 34 mJ with 500-µs pulse duration and 150-Hz pulse rate was delivered through the hollow-tip fibers in contact with human calcium oxalate monohydrate urinary stones during ex vivo studies. Significant fiber tip burn-back and degradation was observed for bare 150-µm core-diameter fibers. However, hollow steel tip fibers experienced minimal fiber burn-back without compromising stone ablation rates. A simple, robust, compact, and inexpensive hollow fiber tip design was characterized for minimizing distal fiber burn-back during the TFL lithotripsy. Although an increase in stone retropulsion was observed, potential integration of the hollow fiber tip into a stone basket may provide rapid stone vaporization, while minimizing retropulsion.

Detachable fiber optic tips for use in thulium fiber laser lithotripsy.

The thulium fiber laser (TFL) has recently been proposed as an alternative to the Holmium:YAG (Ho:YAG) laser for lithotripsy. The TFL's Gaussian spatial beam profile provides higher power transmission through smaller optical fibers with reduced proximal fiber tip damage, and improved saline irrigation and flexibility through the ureteroscope. However, distal fiber tip damage may still occur during stone fragmentation, resulting in disposal of the entire fiber after the procedure. A novel design for a short, detachable, distal fiber tip that can fit into an ureteroscope's working channel is proposed. A prototype, twist-lock, spring-loaded mechanism was constructed using micromachining methods, mating a 150-µm-core trunk fiber to 300-µm-core fiber tip. Optical transmission measuring 80% was observed using a 30-mJ pulse energy and 500-µs pulse duration. Ex vivo human calcium oxalate monohydrate urinary stones were vaporized at an average rate of 187 µg/s using 20-Hz modulated, 50% duty cycle 5 pulse packets. The highest stone ablation rates corresponded to the highest fiber tip degradation, thus providing motivation for use of detachable and disposable distal fiber tips during lithotripsy. The 1-mm outer-diameter prototype also functioned comparable to previously tested tapered fiber tips.

Fiber-optic manipulation of urinary stone phantoms using holmium:YAG and thulium fiber lasers.

Fiber-optic attraction of urinary stones during laser lithotripsy may be exploited to manipulate stone fragments inside the urinary tract without mechanical grasping tools, saving the urologist time and space in the ureteroscope working channel. We compare thulium fiber laser (TFL) high pulse rate/low pulse energy operation to conventional holmium:YAG low pulse rate/high pulse energy operation for fiber-optic suctioning of plaster-of-paris (PoP) stone phantoms. A TFL (wavelength of 1908 nm, pulse energy of 35 mJ, pulse duration of 500 µs, and pulse rate of 10 to 350 Hz) and a holmium laser (wavelength of 2120 nm, pulse energy of 35 to 360 mJ, pulse duration of 300 µs, and pulse rate of 20 Hz) were tested using 270-µm-core optical fibers. A peak drag speed of ~2.5 mm/s was measured for both TFL (35 mJ and 150 to 250 Hz) and holmium laser (210 mJ and 20 Hz). Particle image velocimetry and thermal imaging were used to track water flow for all parameters. Fiber-optic suctioning of urinary stone phantoms is feasible. TFL operation at high pulse rates/low pulse energies is preferable to holmium operation at low pulse rates/high pulse energies for rapid and smooth stone pulling. With further development, this novel technique may be useful for manipulating stone fragments in the urinary tract.

Enhanced thulium fiber laser lithotripsy using micro-pulse train modulation.

The thulium fiber laser (TFL) is currently being studied as an alternative to the conventional holmium:YAG (Ho:YAG) laser for lithotripsy. The diode-pumped TFL may be electronically modulated to operate with variable parameters (e.g., pulse rate, pulse duration, and duty cycle) for studying the influence of pulse train mode on stone ablation rates. The TFL under study was operated at 1908 nm, 35-mJ pulse energy, and 500-µs pulse duration, in a train of 5 micro-pulses, with macro-pulse rates of 10 Hz, compared with conventional TFL operation at 10 to 50 Hz. TFL energy was delivered through 100-µm-core fibers in contact with human uric acid (UA) and calcium oxalate monohydrate (COM) stones. Mass removal rates, optical coherence tomography, and light microscopy were used to analyze the ablation craters. Stone retropulsion and fiber tip degradation studies also were conducted for these laser parameters. TFL operation in micro-pulse train (MPT) mode resulted in a factor of two increase in the ablation rate of 414 ± 94 µg/s and 122 ± 24 µg/s for the UA and COM stones, respectively, compared to 182 ± 69 µg/s and 60 ± 14 µg/s with standard pulse trains delivered at 50 Hz (P<0.05). Stone retropulsion remained minimal (<2 mm after 1200 pulses) for both pulse modes. Fiber burnback was significant for both pulse modes and was higher for COM stones than UA stones. TFL operation in MPT mode results in increased stone ablation rates which, with further optimization, may approach levels comparable to Ho:YAG laser lithotripsy in the clinic.

Comparison of holmium:YAG and thulium fiber laser lithotripsy: ablation thresholds, ablation rates, and retropulsion effects.

The holmium:YAG (Ho:YAG) laser lithotriptor is capable of operating at high pulse energies, but efficient operation is limited to low pulse rates (∼10 Hz) during lithotripsy. On the contrary, the thulium fiber laser (TFL) is limited to low pulse energies, but can operate efficiently at high pulse rates (up to 1000 Hz). This study compares stone ablation threshold, ablation rate, and retropulsion for the two different Ho:YAG and TFL operation modes. The TFL (λ = 1908 nm) was operated with pulse energies of 5 to 35 mJ, 500-µs pulse duration, and pulse rates of 10 to 400 Hz. The Ho:YAG laser (λ = 2120 nm) was operated with pulse energies of 30 to 550 mJ, 350-µs pulse duration, and a pulse rate of 10 Hz. Laser energy was delivered through 200- and 270-µm-core optical fibers in contact mode with human calcium oxalate monohydrate (COM) stones for ablation studies and plaster-of-Paris stone phantoms for retropulsion studies. The COM stone ablation threshold for Ho:YAG and TFL measured 82.6 and 20.8 J∕cm(2), respectively. Stone retropulsion with the Ho:YAG laser linearly increased with pulse energy. Retropulsion with TFL was minimal at pulse rates less than 150 Hz, then rapidly increased at higher pulse rates. For minimal stone retropulsion, Ho:YAG operation at pulse energies less than 175 mJ at 10 Hz and TFL operation at 35 mJ at 100 Hz is recommended, with both lasers producing comparable ablation rates. Further development of a TFL operating with both high pulse energies of 100 to 200 mJ and high pulse rates of 100 to 150 Hz may also provide an alternative to the Ho:YAG laser for higher ablation rates, when retropulsion is not a primary concern.

Holmium:YAG (lambda = 2,120 nm) versus thulium fiber (lambda = 1,908 nm) laser lithotripsy.

INTRODUCTION: The holmium:YAG laser is currently the most common laser lithotripter. However, recent experimental studies have demonstrated that the thulium fiber laser is also capable of vaporizing urinary stones. The high-temperature water absorption coefficient for the thulium wavelength (mu(a) = 160 cm(-1) at lambda = 1,908 nm) is significantly higher than for the holmium wavelength (mu(a) = 28 cm(-1) at lambda = 2,120 nm). We hypothesize that this should translate into more efficient laser lithotripsy using the thulium fiber laser. This study directly compares stone vaporization rates for holmium and thulium fiber lasers. METHODS: Holmium laser radiation pulsed at 3 Hz with 70 mJ pulse energy and 220 microseconds pulse duration was delivered through a 100-microm-core silica fiber to human uric acid (UA) and calcium oxalate monohydrate (COM) stones, ex vivo (n = 10 each). Thulium fiber laser radiation pulsed at 10 Hz with 70 mJ pulse energy and 1-millisecond pulse duration was also delivered through a 100-microm fiber for the same sets of 10 stones each. RESULTS: For the same number of pulses and total energy (126 J) delivered to each stone, the mass loss averaged 2.4+/-0.6 mg (UA) and 0.7+/-0.2 mg (COM) for the holmium laser and 12.6+/-2.5 mg (UA) and 6.8+/-1.7 (COM) for the thulium fiber laser. CONCLUSIONS: UA and COM stone vaporization rates for the thulium fiber laser averaged 5-10 times higher than for the holmium laser at 70 mJ pulse energies. With further development, the thulium fiber laser may represent an alternative to the conventional holmium laser for more efficient laser lithotripsy.

Thulium fiber laser lithotripsy using tapered fibers.

INTRODUCTION: The Thulium fiber laser has recently been tested as a potential alternative to the Holmium:YAG laser for lithotripsy. This study explores use of a short taper for expanding the Thulium fiber laser beam at the distal tip of a small-core fiber. METHODS: Thulium fiber laser radiation with a wavelength of 1,908 nm, 10 Hz pulse rate, 70 mJ pulse energy, and 1-millisecond pulse duration was delivered through a 2-m-length fiber with 150-microm-core-input-end, 300-microm-core-output-end, and 5-mm-length taper, in contact with human uric acid (UA) and calcium oxalate monohydrate (COM) stones, ex vivo (n = 10 each). Stone mass loss, stone crater depths, fiber transmission losses, fiber burn-back, irrigation rates, and deflection through a flexible ureteroscope were measured for the tapered fiber and compared with conventional fibers. RESULTS: After delivery of 1,800 pulses through the tapered fiber, mass loss measured 12.7+/-2.6 mg for UA and 7.2+/-0.8 mg COM stones, comparable to conventional 100-microm-core fibers (12.6+/-2.5 mg for UA and 6.8+/-1.7 mg for COM stones). No transmission losses or burn-back occurred for the tapered fiber after 36,000 pulses, while a conventional 150-microm fiber experienced significant tip degradation after only 1,800 pulses. High irrigation rates were measured with the tapered fiber inserted through the working port of a flexible ureteroscope without hindering its deflection, mimicking that of a conventional 150 microm fiber. CONCLUSIONS: The short tapered distal fiber tip allows expansion of the laser beam, resulting in decreased fiber tip damage compared to conventional small-core fibers, without compromising fiber bending, stone vaporization efficiency, or irrigation rates.