Measurement of rat and human tissue optical properties for improving the optical detection and visualization of peripheral nerves.

Peripheral nerve damage frequently occurs in challenging surgical cases resulting in high costs and morbidity. Various optical techniques have proven effective in detecting and visually enhancing nerves, demonstrating their translational potential for assisting in nerve-sparing medical procedures. However, there is limited data characterizing the optical properties of nerves in comparison to surrounding tissues, thus limiting the optimization of optical nerve detection systems. To address this gap, the absorption and scattering properties of rat and human nerve, muscle, fat, and tendon were determined from 352-2500 nm. The optical properties highlighted an ideal region in the shortwave infrared for detecting embedded nerves, which remains a significant challenge for optical approaches. A 1000-1700 nm hyperspectral diffuse reflectance imaging system was used to confirm these results and identify optimal wavelengths for nerve imaging contrast in an in vivo rat model. Optimal nerve visualization contrast was achieved using 1190/1100 nm ratiometric imaging and was sustained for nerves embedded under ≥600 µm of fat and muscle. Overall, the results provide valuable insights for optimizing the optical contrast of nerves, including those embedded in tissue, which could lead to improved surgical guidance and nerve-sparing outcomes.

Label-free intraoperative nerve detection and visualization using ratiometric diffuse reflectance spectroscopy.

Iatrogenic nerve injuries contribute significantly to postoperative morbidity across various surgical disciplines and occur in approximately 500,000 cases annually in the US alone. Currently, there are no clinically adopted means to intraoperatively visualize nerves beyond the surgeon's visual assessment. Here, we report a label-free method for nerve detection using diffuse reflectance spectroscopy (DRS). Starting with an in vivo rat model, fiber- and imaging-based DRS independently identified similar wavelengths that provided optimal contrast for nerve identification with an accuracy of 92%. Optical property measurements of rat and human cadaver tissues verify that the source of contrast between nerve and surrounding tissues is largely due to higher scattering in nerve and differences in oxygenated hemoglobin content. Clinical feasibility was demonstrated in patients undergoing thyroidectomies using both probe-based and imaging-based approaches where the nerve were identified with 91% accuracy. Based on our preliminary results, DRS has the potential to both provide surgeons with a label-free, intraoperative means of nerve visualization and reduce the incidence of iatrogenic nerve injuries along with its detrimental complications.

Infrared neural stimulation markedly enhances nerve functionality assessment during nerve monitoring.

In surgical procedures where the risk of accidental nerve damage is prevalent, surgeons commonly use electrical stimulation (ES) during intraoperative nerve monitoring (IONM) to assess a nerve's functional integrity. ES, however, is subject to off-target stimulation and stimulation artifacts disguising the true functionality of the specific target and complicating interpretation. Lacking a stimulation artifact and having a higher degree of spatial specificity, infrared neural stimulation (INS) has the potential to improve upon clinical ES for IONM. Here, we present a direct comparison between clinical ES and INS for IONM performance in an in vivo rat model. The sensitivity of INS surpasses that of ES in detecting partial forms of damage while maintaining a comparable specificity and sensitivity to more complete forms. Without loss in performance, INS is readily compatible with existing clinical nerve monitoring systems. These findings underscore the clinical potential of INS to improve IONM and surgical outcomes.

Visualizing the lipid dynamics role in infrared neural stimulation using stimulated Raman scattering.

Infrared neural stimulation (INS) uses pulsed infrared light to yield label-free neural stimulation with broad experimental and translational utility. Despite its robust demonstration, INS's mechanistic and biophysical underpinnings have been the subject of debate for more than a decade. The role of lipid membrane thermodynamics appears to play an important role in how fast IR-mediated heating nonspecifically drives action potential generation. Direct observation of lipid membrane dynamics during INS remains to be shown in a live neural model system. We used hyperspectral stimulated Raman scattering microscopy to study biochemical signatures of high-speed vibrational dynamics underlying INS in a live neural cell culture model. The findings suggest that lipid bilayer structural changes occur during INS in vitro in NG108-15 neuroglioma cells. Lipid-specific signatures of cell stimulated Raman scattering spectra varied with stimulation energy and radiation exposure. The spectroscopic observations agree with high-speed ratiometric fluorescence imaging of a conventional lipophilic membrane structure reporter, 4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)pyridinium hydroxide. The findings support the hypothesis that INS causes changes in the lipid membrane of neural cells by changing the lipid membrane packing order. This work highlights the potential of hyperspectral stimulated Raman scattering as a method to safely study biophysical and biochemical dynamics in live cells.

Identifying optimal parameters for infrared neural stimulation in the peripheral nervous system.

Significance: Infrared neural stimulation (INS) utilizes pulsed infrared light to selectively elicit neural activity without exogenous compounds. Despite its versatility in a broad range of biomedical applications, no comprehensive comparison of factors pertaining to the efficacy and safety of INS such as wavelength, radiant exposure, and optical spot size exists in the literature. Aim: Here, we evaluate these parameters using three of the wavelengths commonly used for INS, 1450 nm, 1875 nm, and 2120 nm. Approach: In an in vivo rat sciatic nerve preparation, the stimulation threshold and transition rate to 100% activation probability were used to compare the effects of each parameter. Results: The pulsed diode lasers at 1450 nm and 1875 nm had a consistently higher ( ∼ 1.0 J / cm 2 ) stimulation threshold than that of the Ho:YAG laser at 2120 nm ( ∼ 0.7 J / cm 2 ). In addition, the Ho:YAG produced a faster transition rate to 100% activation probability compared to the diode lasers. Our data suggest that the superior performance of the Ho:YAG is a result of the high-intensity microsecond spike at the onset of the pulse. Acute histological evaluation of diode irradiated nerves revealed a safe range of radiant exposures for stimulation. Conclusion: Together, our results identify measures to improve the safety, efficacy, and accessibility of INS technology for research and clinical applications.

Multi-modal nonlinear optical and thermal imaging platform for label-free characterization of biological tissue.

The ability to characterize the combined structural, functional, and thermal properties of biophysically dynamic samples is needed to address critical questions related to tissue structure, physiological dynamics, and disease progression. Towards this, we have developed an imaging platform that enables multiple nonlinear imaging modalities to be combined with thermal imaging on a common sample. Here we demonstrate label-free multimodal imaging of live cells, excised tissues, and live rodent brain models. While potential applications of this technology are wide-ranging, we expect it to be especially useful in addressing biomedical research questions aimed at the biomolecular and biophysical properties of tissue and their physiology.

Asymmetrical bipolar nanosecond electric pulse widths modify bipolar cancellation.

A bipolar (BP) nanosecond electric pulse (nsEP) exposure generates reduced calcium influx compared to a unipolar (UP) nsEP. This attenuated physiological response from a BP nsEP exposure is termed "bipolar cancellation" (BPC). The predominant BP nsEP parameters that induce BPC consist of a positive polarity (↑) front pulse followed by the delivery of a negative polarity (↓) back pulse of equal voltage and width; thereby the duration is twice a UP nsEP exposure. We tested these BPC parameters, and discovered that a BP nsEP with symmetrical pulse widths is not required to generate BPC. For example, our data revealed the physiological response initiated by a ↑900 nsEP exposure can be cancelled by a second pulse that is a third of its duration.  However, we observed a complete loss of BPC from a ↑300 nsEP followed by a ↓900 nsEP exposure. Spatiotemporal analysis revealed these asymmetrical BP nsEP exposures generate distinct local YO-PRO®-1 uptake patterns across the plasma membrane. From these findings, we generated a conceptual model that suggests BPC is a phenomenon balanced by localized charging and discharging events across the membrane.

Wavefront shaping enhanced Raman scattering in a turbid medium.

Spontaneous Raman scattering is a powerful tool for chemical sensing and imaging but suffers from a weak signal. In this Letter, we present an application of adaptive optics to enhance the Raman scattering signal detected through a turbid, optically thick material. This technique utilizes recent advances in wavefront shaping techniques for focusing light through a turbid media and applies them to chemical detection to achieve a signal enhancement with little sacrifice to the overall simplicity of the experimental setup. With this technique, we demonstrate an enhancement in the Raman signal from titanium dioxide particles through a highly scattering material. This technique may pave the way to label-free tracking using the optical memory effect.