Er:YAG laser brain surgery with vascular specific coagulation.

BACKGROUND AND OBJECTIVE: Erbium:yttrium-aluminum-garnet (Er:YAG) laser ablation can effectively resect water-bearing tissues. Application of Er:YAG resection in neurosurgery is complicated by unpredictable bleeding in surgical field. Recently, an integrated theranostic system combining a dual-wavelength laser surgery system using a thulium (Tm) fiber-laser for coagulation and Er:YAG for resection, combined with optical coherence tomography (OCT) guidance was demonstrated for the in vivo resection of tumor tissue. However, lateral thermal spread in the range of 100s of micrometers is common due to lack of vascular specificity using a Tm fiber-laser for coagulation. In this study, a vascular specific ytterbium (Yb) fiber-laser is utilized for enhanced photocoagulation during in vivo neurosurgery improving the precision of Er:YAG tissue resection with minimal lateral thermal spread. METHODS: Mice underwent stereotactic laser surgery with the proposed Yb/Er:YAG dual wavelength vascular specific neurosurgery in vivo. An OCT system (wavelength range 1310 ± 70 nm) and OCT derived angiography  images were used to record cortical images to confirm the coagulation of blood vessels and guide subsequent Er:YAG resection steps. After the laser surgery, mice were killed, and histological analysis was carried out using hematoxylin and eosin staining and Nissl staining to compare the lateral thermal spread with our previously reported Tm/Er:YAG neurosurgery where a continuous wave  Tm fiber-laser was used for coagulation. RESULTS: Coagulation scheme using a Yb fiber-laser allowed stoppage of blood flow in disparately sized blood vessels encountered in the mice brain. Histological analysis of murine brain slices post Yb/Er:YAG laser surgery yielded lower thermal spread compared with Tm/Er:YAG laser surgery, maximizing the efficiency in both hemostasis (blood flow stoppage) and maximizing tissue ablation efficiency with minimal residual thermal damage zone. CONCLUSION: In this study, a vascular specific coagulation scheme with Yb/Er:YAG dual-wavelength surgery is presented for neurosurgery. Additionally, Yb/Er:YAG study results are compared with that of a tissue coagulation approach in Tm/Er:YAG surgery previously reported to highlight improved coagulation, reduced nonspecific thermal damage and limited lateral thermal spread. Experimental results suggest that the developed dual-wavelength laser system can effectively resect neural tissues with high localization, minimal lateral thermal spread at the micrometer level while maintaining a bloodless surgical field.

Laser coagulation and hemostasis of large diameter blood vessels: effect of shear stress and flow velocity.

Photocoagulation of blood vessels offers unambiguous advantages to current radiofrequency approaches considering the high specificity of blood absorption at available laser wavelengths (e.g., 532 nm and 1.064 µm). Successful treatment of pediatric vascular lesions, such as port-wine stains requiring microvascular hemostasis, has been documented. Although laser treatments have been successful in smaller diameter blood vessels, photocoagulation of larger sized vessels is less effective. The hypothesis for this study is that a primary limitation in laser coagulation of large diameter blood vessels (500-1000 µm) originates from shear stress gradients associated with higher flow velocities along with temperature-dependent viscosity changes. Laser (1.07 µm) coagulation of blood vessels was tested in the chicken chorio-allantoic membrane (CAM). A finite element model is developed that includes hypothetical limitations in laser coagulation during irradiation. A protocol to specify laser dosimetry is derived from OCT imaging and angiography observations as well as finite element model results. Laser dosimetry is applied in the CAM model to test the experimental hypothesis that blood shear stress and flow velocity are important parameters for laser coagulation and hemostasis of large diameter blood vessels (500-1000 µm). Our experimental results suggest that shear stress and flow velocity are fundamental in the coagulation of large diameter blood vessels (500-1000 µm). Laser dosimetry is proposed and demonstrated for successful coagulation and hemostasis of large diameter CAM blood vessels.

Differentiation of Brain Tumor Microvasculature From Normal Vessels Using Optical Coherence Angiography.

BACKGROUND AND OBJECTIVES: Despite rapid advances and discoveries in medical imaging, monitoring therapeutic efficacy for malignant gliomas and monitoring tumor vasculature remains problematic. The purpose of this study is to utilize optical coherence angiography for vasculature characterization inside and surrounding brain tumors in a murine xenograft brain tumor model. Features included in our analysis include fractional blood volume, vessel tortuosity, diameter, orientation, and directionality. STUDY DESIGN/MATERIALS AND METHODS: In this study, five tumorous mice models at 4 weeks of age were imaged. Human glioblastoma cells were injected into the brain and allowed to grow for 4 weeks and then imaged using optical coherence tomography. RESULTS: Results suggest that blood vessels outside the tumor contain a greater fractional blood volume as compared with vessels inside the tumor. Vessels inside the tumor are more tortuous as compared with those outside the tumor. Results indicate that vessels near the tumor margin are directed inward towards the tumor while normal vessels show a more random orientation. CONCLUSION: Quantification of vascular microenvironments in brain gliomas can provide functional vascular parameters to aid various diagnostic and therapeutic studies. © 2021 Wiley Periodicals LLC.

B.2.16 is a non-lethal modifier of the Dark82 mosaic eye phenotype in Drosophila melanogaster.

Genetic screens have been used to identify genes involved in the regulation of different biological processes. We identified growth mutants in a Flp/FRT screen using the Drosophila melanogaster eye to identify conditional regulators of cell growth and cell division. One mutant identified from this screen, B.2.16, was mapped and characterized by researchers in undergraduate genetics labs as part of the Fly-CURE. We find that B.2.16 is a non-lethal genetic modifier of the Dark82 mosaic eye phenotype.

Laser brain cancer surgery in a xenograft model guided by optical coherence tomography.

Higher precision surgical devices are needed for tumor resections near critical brain structures. The goal of this study is to demonstrate feasibility of a system capable of precise and bloodless tumor ablation. An image-guided laser surgical system is presented for excision of brain tumors in vivo in a murine xenograft model. The system combines optical coherence tomography (OCT) guidance with surgical lasers for high-precision tumor ablation (Er:YAG) and microcirculation coagulation (Thulium (Tm) fiber laser). Methods: A fluorescent human glioblastoma cell line was injected into mice and allowed to grow four weeks. Craniotomies were performed and tumors were imaged with confocal fluorescence microscopy. The mice were subsequently OCT imaged prior, during and after laser coagulation and/or ablation. The prior OCT images were used to compute three-dimensional tumor margin and angiography images, which guided the coagulation and ablation steps. Histology of the treated regions was then compared to post-treatment OCT images. Results: Tumor sizing based on OCT margin detection matched histology to within experimental error. Although fluorescence microscopy imaging showed the tumors were collocated with OCT imaging, margin assessment using confocal microscopy failed to see the extent of the tumor beyond ~ 250 µm in depth, as verified by OCT and histology. The two-laser approach to surgery utilizing Tm wavelength for coagulation and Er:YAG for ablation yielded bloodless resection of tumor regions with minimal residual damage as seen in histology. Conclusion: Precise and bloodless tumor resection under OCT image guidance is demonstrated in the murine xenograft brain cancer model. Tumor margins and vasculature are accurately made visible without need for exogenous contrast agents.

A human skin high-throughput formulation screening method using a model hydrophilic drug.

Franz cell (FC) experiments in topical and transdermal drug development represent the gold standard in vitro method but require a relatively high quantity of human skin, are low-throughput, and are time-consuming to perform. To address these issues, we studied a micro-well plate-based screening method for permeability and retention that could enable the direct screening of large numbers of formulations simultaneously across human skin. Using freshly excised dermatomed human skin modified to reflect poor barrier function and a model hydrophilic compound, Sulforhodamine B (SRB), FC permeation and retention quantification was compared to the 96-well high-throughput system (HTS). The skin was analyzed using 2-photon microscopy to determine the drug distribution within the skin. A screen of 15 different formulations in triplicate in a single piece of human skin, using full factorial design was then conducted. Permeability of SRB across the skin as well as the drug distribution profile of SRB retained in the skin were similar for the FC and HTS system. The influence of different excipients on drug retention was observed in the full factorial formulation screen. The HTS method is promising for the investigation of large numbers of formulations and the influence of formulations changes in skin retention of drug.

In situ process monitoring in selective laser sintering using optical coherence tomography.

Selective laser sintering (SLS) is an efficient process in additive manufacturing that enables rapid part production from computer-based designs. However, SLS is limited by its notable lack of in-situ process monitoring when compared to other manufacturing processes. We report the incorporation of optical coherence tomography into an SLS system in detail and demonstrate access to surface and sub-surface features. Video frame rate cross-sectional imaging reveals areas of sintering uniformity and areas of excessive heat error with high temporal resolution. We propose a set of image processing techniques for SLS process monitoring with OCT and report the limitations and obstacles for further OCT integration with SLS systems.

Optical coherence tomography image-guided smart laser knife for surgery.

BACKGROUND AND OBJECTIVE: Surgical oncology can benefit from specialized tools that enhance imaging and enable precise cutting and removal of tissue without damage to adjacent structures. The combination of high-resolution, fast optical coherence tomography (OCT) co-aligned with a nanosecond pulsed thulium (Tm) laser offers advantages over conventional surgical laser systems. Tm lasers provide superior beam quality, high volumetric tissue removal rates with minimal residual thermal footprint in tissue, enabling a reduction in unwanted damage to delicate adjacent sub-surface structures such as nerves or micro-vessels. We investigated such a combined Tm/OCT system with co-aligned imaging and cutting beams-a configuration we call a "smart laser knife." METHODS: A blow-off model that considers absorption coefficients and beam delivery systems was utilized to predict Tm cut depth, tissue removal rate and spatial distribution of residual thermal injury. Experiments were performed to verify the volumetric removal rate predicted by the model as a function of average power. A bench-top, combined Tm/OCT system was constructed using a 15W 1940 nm nanosecond pulsed Tm fiber laser (500 µJ pulse energy, 100 ns pulse duration, 30 kHz repetition rate) for removing tissue and a swept source laser (1310 ± 70 nm, 100 kHz sweep rate) for OCT imaging. Tissue phantoms were used to demonstrate precise surgery with blood vessel avoidance. Depth imaging informed cutting/removal of targeted tissue structures by the Tm laser was performed. RESULTS: Laser cutting was accomplished around and above phantom blood vessels while avoiding damage to vessel walls. A tissue removal rate of 5.5 mm3 /sec was achieved experimentally, in comparison to the model prediction of approximately 6 mm3 /sec. CONCLUSION: We describe a system that combines OCT and laser tissue modification with a Tm laser. Simulation results of the tissue removal rate using a simple model, as a function of average power, are in good agreement with experimental results using tissue phantoms. Lasers Surg. Med. 50:202-212, 2018. © 2017 Wiley Periodicals, Inc.

Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion.

Occlusions in single cortical microvessels lead to a reduction in oxygen supply, but this decrement has not been able to be quantified in three dimensions at the level of individual vessels using a single instrument. We demonstrate a combined optical system using two-photon phosphorescence lifetime and fluorescence microscopy (2PLM) to characterize the partial pressure of oxygen (pO2) in single descending cortical arterioles in the mouse brain before and after generating a targeted photothrombotic occlusion. Integrated real-time Laser Speckle Contrast Imaging (LSCI) provides wide-field perfusion maps that are used to monitor and guide the occlusion process while 2PLM maps changes in intravascular oxygen tension. We present the technique's utility in highlighting the effects of vascular networking on the residual intravascular oxygen tensions measured after occlusion in three dimensions.

Depth-resolved blood oxygen saturation measurement by dual-wavelength photothermal (DWP) optical coherence tomography.

Non-invasive depth-resolved measurement of hemoglobin oxygen saturation (SaO(2)) levels in discrete blood vessels may have implications for diagnosis and treatment of various pathologies. We introduce a novel Dual-Wavelength Photothermal (DWP) Optical Coherence Tomography (OCT) for non-invasive depth-resolved measurement of SaO(2) levels in a blood vessel phantom. DWP OCT SaO(2) is linearly correlated with blood-gas SaO(2) measurements. We demonstrate 6.3% precision in SaO(2) levels measured a phantom blood vessel using DWP-OCT with 800 and 765 nm excitation wavelengths. Sources of uncertainty in SaO(2) levels measured with DWP-OCT are identified and characterized.

Improved sensitivity for two-photon frequency-domain lifetime measurement.

We demonstrate a method to improve the measurement sensitivity of two-photon frequency-domain lifetime measurements in poor signal to background conditions. This technique uses sinusoidal modulation of the two-photon excitation source and detection of the second harmonic of the modulation frequency that appears in the emission. Additionally, we present the mathematical model which describes how the observed phase shift and amplitude demodulation factor of two-photon phosphorescence emission are related to the phosphorescence lifetime and modulation frequency. We demonstrate the validity of the model by showing the existence of new frequency terms in the phosphorescence emission generated from the quadratic nature of two-photon absorption and by showing that the phase shift and demodulation match theory for all frequency components.

Intra-organ Biodistribution of Gold Nanoparticles Using Intrinsic Two-photon Induced Photoluminescence.

BACKGROUND AND OBJECTIVES: Gold nanoparticles (GNPs) such as gold nanoshells (GNSs) and gold nanorods (GNRs) have been explored in a number of in vitro and in vivo studies as imaging contrast and cancer therapy agents due to their highly desirable spectral and molecular properties. While the organ-level biodistribution of these particles has been reported previously, little is known about the cellular level or intra-organ biodistribution. The objective of this study was to demonstrate the use of intrinsic two-photon induced photoluminescence (TPIP) to study the cellular level biodistribution of GNPs. STUDY DESIGN/MATERIALS AND METHODS: Tumor xenografts were created in twenty-seven male nude mice (Swiss nu/nu) using HCT 116 cells (CCL-247, ATCC, human colorectal cancer cell line). GNSs and GNRs were systemically injected 24 hr. prior to tumor harvesting. A skin flap with the tumor was excised and sectioned as 8 µm thick tissues for imaging GNPs under a custom-built multiphoton microscope. For multiplexed imaging, nuclei, cytoplasm, and blood vessels were demonstrated by hematoxylin and eosin (H&E) staining, YOYO-1 iodide staining and CD31-immunofluorescence staining. RESULTS: Distribution features of GNPs at the tumor site were determined from TPIP images. GNSs and GNRs had a heterogeneous distribution with higher accumulation at the tumor cortex than tumor core. GNPs were also observed in unique patterns surrounding the perivascular region. While most GNSs were confined at the distance of approximately 400 µm inside the tumor edge, GNRs were shown up to 1.5 mm penetration inside the edge. CONCLUSIONS: We have demonstrated the use of TPIP imaging in a multiplexed fashion to image both GNPs and nuclei, cytoplasm, or vasculature simultaneously. We also confirmed that TPIP imaging enabled visualization of GNP distribution patterns within the tumor and other critical organs. These results suggest that direct luminescence-based imaging of metal nanoparticles holds a valuable and promising position in understanding the accumulation kinetics of GNPs. In addition, these techniques will be increasingly important as the use of these particles progress to human clinical trials where standard histopathology techniques are used to analyze their effects.

Two-photon-induced photoluminescence imaging of tumors using near-infrared excited gold nanoshells.

Gold nanoshells (dielectric silica core/gold shell) are a novel class of hybrid metal nanoparticles whose unique optical properties have spawned new applications including more sensitive molecular assays and cancer therapy. We report a new photo-physical property of nanoshells (NS) whereby these particles glow brightly when excited by near-infrared light. We characterized the luminescence brightness of NS, comparing to that of gold nanorods (NR) and fluorescent beads (FB). We find that NS are as bright as NR and 140 times brighter than FB. To demonstrate the potential application of this bright two-photon-induced photoluminescence (TPIP) signal for biological imaging, we imaged the 3D distribution of gold nanoshells targeted to murine tumors.

Microvascular oxygen quantification using two-photon microscopy.

An instrument is demonstrated that is capable of three-dimensional (3D) vasculature imaging and pO(2) quantification with high spatial resolution. The instrument combines two-photon (2P) microscopy with phosphorescence quenching to measure pO(2). The instrument was demonstrated by performing depth-resolved microvascular pO(2) measurements of rat cortical vessels down to 120 microm below the surface. 2P excitation of porphyrin was confirmed, and measured pO(2) values were consistent with previously published data for normoxic and hyperoxic conditions. The ability to perform 3D pO(2) measurements using optical techniques will allow researchers to overcome existing limitations imposed by polarographic electrodes, magnetic resonance techniques, and surface-only pO(2) measurement techniques.