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.

In vivo Raman spectroscopy monitors cervical change during labor.

BACKGROUND: Biochemical cervical change during labor is not well understood, in part, because of a dearth of technologies capable of safely probing the pregnant cervix in vivo. The need for such a technology is 2-fold: (1) to gain a mechanistic understanding of the cervical ripening and dilation process and (2) to provide an objective method for evaluating the cervical state to guide clinical decision-making. Raman spectroscopy demonstrates the potential to meet this need, as it is a noninvasive optical technique that can sensitively detect alterations in tissue components, such as extracellular matrix proteins, lipids, nucleic acids, and blood, which have been previously established to change during the cervical remodeling process. OBJECTIVE: We sought to demonstrate that Raman spectroscopy can longitudinally monitor biochemical changes in the laboring cervix to identify spectral markers of impending parturition. STUDY DESIGN: Overall, 30 pregnant participants undergoing either spontaneous or induced labor were recruited. The Raman spectra were acquired in vivo at 4-hour intervals throughout labor until rupture of membranes using a Raman system with a fiber-optic probe. Linear mixed-effects models were used to determine significant (P<.05) changes in peak intensities or peak ratios as a function of time to delivery in the study population. A nonnegative least-squares biochemical model was used to extract the changing contributions of specific molecule classes over time. RESULTS: We detected multiple biochemical changes during labor, including (1) significant decreases in Raman spectral features associated with collagen and other extracellular matrix proteins (P=.0054) attributed to collagen dispersion, (2) an increase in spectral features associated with blood (P=.0372), and (3) an increase in features indicative of lipid-based molecules (P=.0273). The nonnegative least-squares model revealed a decrease in collagen contribution with time to delivery, an increase in blood contribution, and a change in lipid contribution. CONCLUSION: Our findings have demonstrated that in vivo Raman spectroscopy is sensitive to multiple biochemical remodeling changes in the cervix during labor. Furthermore, in vivo Raman spectroscopy may be a valuable noninvasive tool for objectively evaluating the cervix to potentially guide clinical management of labor.

Dual excitation wavelength system for combined fingerprint and high wavenumber Raman spectroscopy.

A fiber optic probe-based Raman spectroscopy system using a single laser module with two excitation wavelengths, at 680 and 785 nm, has been developed for measuring the fingerprint and high wavenumber regions using a single detector. This system is simpler and less expensive than previously reported configurations of combined fingerprint and high wavenumber Raman systems, and its probe-based implementation facilitates numerous in vivo applications. The high wavenumber region of the Raman spectrum ranges from 2800-3800 cm-1 and contains valuable information corresponding to the molecular vibrations of proteins, lipids, and water, which is complimentary to the biochemical signatures found in the fingerprint region (800-1800 cm-1), which probes DNA, lipids, and proteins. The efficacy of the system is demonstrated by tracking changes in water content in tissue-mimicking phantoms, where Voigtian decomposition of the high wavenumber water peak revealed a correlation between the water content and type of water-tissue interactions in the samples. This dual wavelength system was then used for in vivo assessment of cervical remodeling during mouse pregnancy, a physiologic process with known changes in tissue hydration. The system shows that Raman spectroscopy is sensitive to changes in collagen content in the fingerprint region and hydration state in the high wavenumber region, which was verified using an ex vivo comparison of wet and dry weight. Simultaneous fingerprint and high wavenumber Raman spectroscopy will allow precise in vivo quantification of tissue water content in the high wavenumber region, paired with the high biochemical specificity of the fingerprint region.

Application driven assessment of probe designs for Raman spectroscopy.

In vivo Raman spectroscopy has been utilized for the non-invasive, non-destructive assessment of tissue pathophysiology for a variety of applications largely through the use of fiber optic probes to interface with samples of interest. Fiber optic probes can be designed to optimize the collection of Raman-scattered photons from application-dependent depths, and this critical consideration should be addressed when planning a study. Herein we investigate four distinct probe geometries for sensitivity to superficial and deep signals through a Monte Carlo model that incorporates Raman scattering and fluorescence. Experimental validation using biological tissues was performed to accurately recapitulate in vivo scenarios. Testing in biological tissues agreed with modeled results and revealed that microlens designs had slightly enhanced performance at shallow depths (< 1 mm), whereas all of the beampath-modified designs yielded more signal from deep within tissue. Simulation based on fluence maps generated using ray-tracing in the absence of optical scattering had drastically different results as a function of depth for each probe compared to the biological simulation. The contrast in simulation results between the non-scattering and biological tissue phantoms underscores the importance of considering the optical properties of a given application when designing a fiber optic probe. The model presented here can be easily extended for optimization of entirely novel probe designs prior to fabrication, reducing time and cost while improving data quality.

Development of a visually guided Raman spectroscopy probe for cervical assessment during pregnancy.

Preterm birth (PTB) is the leading cause of neonatal death, however, accurate prediction methods do not exist. Detection of early changes in the cervix, an organ that biochemically remodels to deliver the fetus, has potential to predict PTB risk. Researchers have employed light-based methods to monitor biochemical changes in the cervix during pregnancy, however, these approaches required patients to undergo a speculum examination which many patients find uncomfortable and is not standard practice during prenatal care. Herein, a visually guided optical probe is presented that measures the cervix via introduction by bimanual examination, a procedure that is commonly performed during prenatal visits and labor for tactile monitoring of the cervix. The device incorporates a Raman spectroscopy probe for biochemical monitoring and a camera for visualizing measurement location to ensure it is void of cervical mucus and blood. This probe was tested in 15 patients receiving obstetric and gynecological care, and results acquired with and without a speculum revealed similar spectra, demonstrating that the visually guided probe conserved data quality. Additionally, the majority of patients reported reduced discomfort from the device. In summary, the visual guidance probe successfully measured the cervix while integrating with standard prenatal care, reducing a barrier in clinical translation.