Calciphylaxis is a painful, debilitating, and premorbid condition, which presents as calcified vasculature and soft tissues. Traditional diagnosis of calciphylaxis lesions requires an invasive biopsy, which is destructive, time consuming, and often leads to exacerbation of the condition and infection. Furthermore, it is difficult to find small calcifications within a large wound bed. To address this need, a noninvasive diagnostic tool may help clinicians identify ectopic calcified mineral and determine the disease margin. We propose Raman spectroscopy as a rapid, point-of-care, noninvasive, and label-free technology to detect calciphylaxis mineral. Debrided calciphylactic tissue was collected from six patients and assessed by microcomputed tomography (micro-CT). Micro-CT confirmed extensive deposits in three specimens, which were subsequently examined with Raman spectroscopy. Raman spectra confirmed that deposits were consistent with carbonated apatite, consistent with the literature. Raman spectroscopy shows potential as a noninvasive technique to detect calciphylaxis in a clinical environment.

To enable tissue function-based tumor diagnosis over the large number of existing digital mammography systems worldwide, we propose a cost-effective and robust approach to incorporate tomographic optical tissue characterization with separately acquired digital mammograms. Using a flexible contour-based registration algorithm, we were able to incorporate an independently measured two-dimensional x-ray mammogram as structural priors in a joint optical/x-ray image reconstruction, resulting in improved spatial details in the optical images and robust optical property estimation. We validated this approach with a retrospective clinical study of 67 patients, including 30 malignant and 37 benign cases, and demonstrated that the proposed approach can help to distinguish malignant from solid benign lesions and fibroglandular tissues, with a performance comparable to the approach using spatially coregistered optical/x-ray measurements.

Optical clearing, in combination with recently developed optical imaging techniques, enables visualization and acquisition of high-resolution, three-dimensional images of biological structures deep within the tissue. Many different approaches can be used to reduce light absorption and scattering within the tissue, but there is a paucity of research on the quantification of clearing efficacy. With the use of a custom-made spectroscopy system, we developed a way to quantify the quality of clearing in biological tissue and applied it to the mouse brain. Three clearing techniques were compared: BABB (1:2 mixture of benzyl alcohol and benzyl benzoate, also known as Murray’s clear), pBABB (peroxide BABB, a modification of BABB which includes the use of hydrogen peroxide), and passive CLARITY. We found that BABB and pBABB produced the highest degree of optical clearing. Furthermore, the approach allows regional measurement of light attenuation to be performed, and our results show that light is most attenuated in regions with high lipid content. We provide a way to choose between the multiple clearing protocols available, and it could prove useful for evaluating images that are acquired with cleared tissues.

The use of fluorescence video imaging to guide surgery is rapidly expanding, and improvements in camera readout dynamic range have not matched display capabilities. Logarithmic intensity compression is a fast, single-step mapping technique that can map the useable dynamic range of high-bit fluorescence images onto the typical 8-bit display and potentially be a variable dynamic contrast enhancement tool. We demonstrate a ∼4.6  times improvement in image quality quantified by image entropy and a dynamic range reduction by a factor of ∼380 by the use of log-compression tools in processing in vivo fluorescence images.

Oxygen supplementation [hyperoxia, increased fraction of inspired oxygen (FiO₂)] is an indispensable treatment in the intensive care unit for patients in respiratory failure. Like other treatments or drugs, hyperoxia has a risk-benefit profile that guides its clinical use. While hyperoxia is known to damage respiratory epithelium, it is unknown if damage can result in impaired capacity to generate cilia-driven fluid flow. Here, we demonstrate that quantifying cilia-driven fluid flow velocities in the sub-100 μm/s regime (sub-0.25 in./min regime) reveals hyperoxia-mediated damage to the capacity of ciliated respiratory mucosa to generate directional flow. Flow quantification was performed using particle tracking velocimetry optical coherence tomography (PTV-OCT) in ex vivo mouse trachea. The ability of PTV-OCT to detect biomedically relevant flow perturbations in the sub-100 μm/s regime was validated by quantifying temperature- and drug-mediated modulation of flow performance in ex vivo mouse trachea. Overall, PTV-OCT imaging of cilia-driven fluid flow in ex vivo mouse trachea is a powerful and straightforward approach for studying factors that modulate and damage mammalian respiratory ciliary physiology.

Single-cell micro-Raman spectroscopy has been applied to explore cell differentiation in single, live, and malignant cells from two tumor cell lines. The spectra of differentiated cells exhibit substantial enhancement primarily in the intensities of protein peaks with concomitant decrease in intensities of O─P─O asymmetric stretching peaks in DNA/RNA. Principal component analyses show that the spectral score of differentiated cells tends to asymptotically approach that of spectra obtained from normal neural stem cells/progenitors. This lends credence to the notion that the observed spectral changes are specific to differentiation, since upon differentiation, malignant cells become less malignant and tend toward benignity.

Over two decades, the Monte Carlo technique has become a gold standard in simulation of light propagation in turbid media, including biotissues. Technological solutions provide further advances of this technique. The Intel Xeon Phi coprocessor is a new type of accelerator for highly parallel general purpose computing, which allows execution of a wide range of applications without substantial code modification. We present a technical approach of porting our previously developed Monte Carlo (MC) code for simulation of light transport in tissues to the Intel Xeon Phi coprocessor. We show that employing the accelerator allows reducing computational time of MC simulation and obtaining simulation speed-up comparable to GPU. We demonstrate the performance of the developed code for simulation of light transport in the human head and determination of the measurement volume in near-infrared spectroscopy brain sensing.

Skin lesions are commonly treated using laser heating. However, the introduction of new devices into clinical practice requires evaluation of their performance. This study presents the application of optical phantoms for assessment of a newly developed 975-nm pulsed diode laser system for dermatological purposes. Such phantoms closely mimic the absorption and scattering of real human skin (although not precisely in relation to thermal conductivity and capacitance); thus, they can be used as substitutes for human skin for approximate evaluation of laser heating efficiency in an almost real environment. Thermographic imaging was applied to measure the spatial and temporal temperature distributions on the surface of laser-irradiated phantoms. The study yielded results of heating with regard to phantom thickness and absorption, as well as laser settings. The methodology developed can be used in practice for preclinical evaluations of laser treatment for dermatology.

Optical coherence tomography (OCT) has become a standard tool in ophthalmology clinics for diagnosing many retinal diseases. Nonetheless, the technical and clinical communities still lack a standardized phantom that could aid in evaluating and normalizing the many protocols and systems used for diagnosis. Existing retinal phantoms are able to mimic the thickness and scattering properties of the retinal layers but are unable to model the morphology of the foveal pit, particularly the tapering of the retinal layers. This work demonstrates a new fabrication procedure that is capable of reliably and consistently replicating the shape and tapered appearance of the retinal layers near the foveal pit using a combination of spin-coating and replica molding. We characterize the effects of using different mold sizes which enable us to achieve a range of pit dimensions. We also present a modified procedure to replicate two diseased states of the retinal tissue, such as retinal detachment and dry aged-related macular degeneration. The ability to create an anatomically correct foveal pit for healthy and disease-mimicking phantoms will allow for a new standard better suited for intra- and inter-system evaluation and for improved comparison of retinal segmentation algorithms.

We demonstrate that information on “intrinsic” anisotropies of fluorescence originating from preferential orientation/organization of fluorophore molecules can be probed using a Mueller matrix of fluorescence. For this purpose, we have developed a simplified model to decouple and separately quantify the depolarization property and the intrinsic anisotropy properties of fluorescence from the experimentally measured fluorescence Mueller matrix. Unlike the traditionally defined fluorescence anisotropy parameter, the Mueller matrix-derived fluorescence polarization metrics, namely, fluorescence diattenuation and polarizance parameters, exclusively deal with the intrinsic anisotropies of fluorescence. The utility of these newly derived fluorescence polarimetry parameters is demonstrated on model systems exhibiting multiple polarimetry effects, and an interesting example is illustrated on biomedically important fluorophores, collagen.

The lung is one of the most common sites of metastases, with approximately 50% of patients with extrathoracic cancer exhibiting pulmonary metastases. Correct identification of the metastatic status of a lung lesion is vital to therapeutic planning and better prognosis. However, currently available diagnostic techniques, such as conventional radiography and low dose computed tomography (LDCT), may fail to identify metastatic lesions. Alternative techniques such as Raman spectroscopy (RS) are hence being extensively explored for correct diagnosis of metastasis. The current ex vivo study aims to evaluate the ability of a fiber optic-based Raman system to distinguish breast cancer metastasis in lung from primary breast and lung tumor in animal models. In this study, spectra were acquired from normal breast, primary breast tumor, normal lung, primary lung tumor, and breast cancer metastasis in lung tissues and analyzed using principal component analysis and principal component-linear discriminant analysis. Breast cancer metastasis in lung could be classified with 71% classification efficiency. Approximately 6% breast metastasis spectra were misclassified with breast tumor, probably due to the presence of breast cancer cells in metastasized lungs. Test prediction results show 64% correct prediction of breast metastasis, while 13% breast metastasis spectra were wrongly predicted as breast tumor, suggesting the possible influence of breast cancer cells. Thus, findings of this study, the first of such explorations, demonstrate the potential of RS in classifying breast metastasis in lungs from primary lung and primary breast tumor. Prospective evaluation on a larger cohort with better multivariate analysis, combined with LDCT and recently developed real-time in vivo probes, RS can play a significant role in nonsurgical screening of lesions, which can lead to individualized therapeutic regimes and improved prognoses.

A polarimetric second harmonic generation (SHG) microscope was used to analyze the dependence between polarization and SHG signal from collagen-based samples. A theoretical model was also developed to investigate the SHG intensity as a function of different polarization states for a set of quasiparallel fibers. Numerical simulations were compared to experimental SHG intensity values and a fairly good agreement was found. Linear polarized light produced periodical changes in the emitted SHG signal with a maximum of intensity corresponding to polarization parallel to the main orientation of the fibers, regardless the ratio of hyperpolarizabilities, ρ. A similar behavior was found for elliptical states located along a vertical meridian on the Poincaré sphere (i.e., null azimuth) although the modulation of the SHG signal was different. Our numerical calculations described a dramatic change in this regular trend when ρ changed from positive to negative values. Moreover, we provide an experimental method (based on the analysis of the modulation of the SHG signal) to determine the value of the ratio ρ and, consequently, to obtain information about the internal organization of the collagen fibers.

We present a dual-modality system for both structural and molecular cell imaging based on coregistered quantitative phase imaging (QPI) and photoacoustic microscopy (PAM). The QPI system was based on off-axis holography, whereas the PAM system comprised a sinusoidally modulated optical source for excitation and a narrow-band low profile and low-cost ring ultrasonic transducer for detection. This approach facilitated a simple confocal alignment of the excitation beams of both modalities and the ultrasonic detector. This system was demonstrated by imaging endogenous molecules in red blood cells (RBCs) as well as by imaging exogenous molecular labels on cancer cells using gold nanoparticles (GNPs) functionalized to target epidermal growth factor receptor. QPI provided high resolution imaging of the cellular structures while PAM provided molecular contrast. This dual-modality microscopy method can potentially be implemented as a compact and low cost cellular diagnostic assay.

Our first step to adapt our recently developed noncontact diffuse correlation tomography (ncDCT) system for three-dimensional (3-D) imaging of blood flow distribution in human breast tumors is reported. A commercial 3-D camera was used to obtain breast surface geometry, which was then converted to a solid volume mesh. An ncDCT probe scanned over a region of interest on the mesh surface and the measured boundary data were combined with a finite element framework for 3-D image reconstruction of blood flow distribution. This technique was tested in computer simulations and in vivo human breasts with low-grade carcinoma. Results from computer simulations suggest that relatively high accuracy can be achieved when the entire tumor is within the sensitive region of diffuse light. Image reconstruction with a priori knowledge of the tumor volume and location can significantly improve the accuracy in recovery of tumor blood flow contrasts. In vivo imaging results from two breast carcinomas show higher average blood flow contrasts (5.9- and 10.9-fold) in the tumor regions compared to the surrounding tissues, which are comparable with previous findings using diffuse correlation spectroscopy. The ncDCT system has the potential to image blood flow distributions in soft and vulnerable tissues without distorting tissue hemodynamics.

Conventional ultrasound (US) and photoacoustic (PA) multimodality imaging require the use of a US pulse for US data acquisition and a laser pulse for PA data acquisition. We propose a method for concurrent US and PA data acquisition with a single-laser pulse. A light-absorbing multilayer film that can generate a US pulse based on the thermoelastic effect is used. The selection of appropriate layer thickness, interlayer spacing, and absorption coefficient allows the spectral characteristics of the generated US signal to be adjusted so that it does not overlap with the spectrum of the PA signal generated by the light transmitting through the layer. Thus, the US signal and the PA signal can be generated, received, and separated by using a single-laser pulse combined with spectral filtering. This method is demonstrated using a multilayer film that generates US signals with a center frequency of 24.2 MHz and fractional bandwidth of 26.8%. The synthetic-aperture focusing technique is applied to improve the lateral resolution and the signal-to-noise ratio. A cyst-like phantom and a film phantom were used to demonstrate the feasibility of this method of concurrent PA-US imaging using single-laser pulses.

Precise device guidance is important for interventional procedures in many different clinical fields including fetal medicine, regional anesthesia, interventional pain management, and interventional oncology. While ultrasound is widely used in clinical practice for real-time guidance, the image contrast that it provides can be insufficient for visualizing tissue structures such as blood vessels, nerves, and tumors. This study was centered on the development of a photoacoustic imaging system for interventional procedures that delivered excitation light in the ranges of 750 to 900 nm and 1150 to 1300 nm, with an optical fiber positioned in a needle cannula. Coregistered B-mode ultrasound images were obtained. The system, which was based on a commercial ultrasound imaging scanner, has an axial resolution in the vicinity of 100 μm and a submillimeter, depth-dependent lateral resolution. Using a tissue phantom and 800 nm excitation light, a simulated blood vessel could be visualized at a maximum distance of 15 mm from the needle tip. Spectroscopic contrast for hemoglobin and lipids was observed with ex vivo tissue samples, with photoacoustic signal maxima consistent with the respective optical absorption spectra. The potential for further optimization of the system is discussed.

Fluorescent labels are well suited as tracers for cancer drug monitoring. Identifying cellular target regions of these drugs with a high resolution is important to assess the working principle of a drug. We investigate the applications of label-free nonresonant four-wave mixing (NR-FWM) microscopy in biological imaging in combination with fluorescence imaging of fluorescently labeled cancer drugs. Results from human A431 tumor cells with stained nuclei and incubated with IRdye 800CW labeled cancer drug cetuximab targeting epidermal growth factor receptor at the cell membrane show that NR-FWM is well suited for cellular imaging. A comparison of vibrationally nonresonant FWM imaging with vibrational resonant coherent anti-Stokes Raman scattering signals revealed nearly identical qualitative information in cellular imaging. NR-FWM is also suitable for tumor tissue imaging in combination with fluorescence imaging of IRdye 800CW labeled, human epidermal growth factor 2 targeting cancer drug pertuzumab and provides additional information over transmission microscopy.

Cerenkov luminescence tomography (CLT) is a promising tool that enables three-dimensional noninvasive in vivo detection of radiopharmaceuticals. Conventionally, multispectral information and diffusion theory were introduced to achieve whole-body tomographic reconstruction. However, the diffusion theory inevitably causes systematic error in blue bands of the electromagnetic spectrum due to high-tissue absorption, and CL has a blue-weighted broad spectrum. Therefore, it is challenging to improve the accuracy of CLT. The performance of the n-order simplified spherical harmonics approximation (SPn) in different spectra is evaluated, and a multispectral hybrid CLT based on the combination of different SPn models is proposed to handle the Cerenkov photon transport problem in complex media. The in vivo xenograft experiment shows that this approach can effectively improve the quality and accuracy of the reconstructed light source. We believe that the new reconstruction method will advance the development of CLT for more in vivo imaging applications.

Ytterbium (Yb³⁺)-sensitized upconverting nanoparticles (UCNPs) are excited at 975 nm causing relatively high absorption in tissue. A new type of UCNPs with neodymium (Nd³⁺) and Yb³⁺ codoping is excitable at a 808-nm wavelength. At this wavelength, the tissue absorption is lower. Here we quantify, both experimentally and theoretically, to what extent Nd³⁺-doped UCNPs will provide an increased signal at larger depths in tissue compared to conventional 975-nm excited UCNPs.

Laser speckle contrast analysis (LASCA) is an established optical technique for accurate widefield visualization of relative blood perfusion when no or minimal scattering from static tissue elements is present, as demonstrated, for example, in LASCA imaging of the exposed cortex. However, when LASCA is applied to diagnosis of burn wounds, light is backscattered from both moving blood and static burn scatterers, and thus the spatial speckle contrast includes both perfusion and nonperfusion components and cannot be straightforwardly associated to blood flow. We extract from speckle contrast images of burn wounds the nonperfusion (static) component and discover that it conveys useful information on the ratio of static-to-dynamic scattering composition of the wound, enabling identification of burns of different depth in a porcine model in vivo within the first 48 h postburn. Our findings suggest that relative changes in the static-to-dynamic scattering composition of burns can dominate relative changes in blood flow for burns of different severity. Unlike conventional LASCA systems that employ scientific or industrial-grade cameras, our LASCA system is realized here using a camera phone, showing the potential to enable LASCA-based burn diagnosis with a simple imager.

Recently, a supramolecular model was developed for predicting striated skeletal muscle intensity profiles obtained by label-free second harmonic generation (SHG) microscopy. This model allows for a quantitative determination of the length of the thick filament antiparallel range or M-band (M), and results in M=0.12  μm for single-band intensity profiles when fixing the A-band length (A) to A=1.6  μm, a value originating from electron microscopy (EM) observations. Using simulations and experimental data sets, we showed that the objective numerical aperture (NA) and the refractive index (RI) mismatch (Δn=n_2ω−n_ω) between the illumination wave (ω) and the second harmonic wave (2ω) severely affect the simulated sarcomere intensity profiles. Therefore, our recovered filament lengths did not match with those observed by EM. For an RI mismatch of Δn=0.02 and a moderate illumination NA of 0.8, analysis of single-band SHG intensity profiles with freely adjustable A- and M-band sizes yielded A=1.40±0.04  μm and M=0.07±0.05  μm for skeletal muscle. These lower than expected values were rationalized in terms of the myosin density distribution along the myosin thick filament axis. Our data provided new and practical insights for the application of the supramolecular model to study SHG intensity profiles in striated muscle.

With its precise, sensitive, and nondestructive features, spectral unmixing-based fluorescence resonance energy transfer (FRET) microscopy has been widely applied to visualize intracellular biological events. In this report, we set up a spectral wide-field microscopic FRET imaging system by integrating a varispec liquid crystal tunable filter into a wide-field microscope for quantitative FRET measurement in living cells. We implemented a representative emission-spectral unmixing-based FRET measurement method on this platform to simultaneously acquire pixel-to-pixel images of both FRET efficiency (E) and acceptor-to-donor concentration ratio (R_C) in living HepG2 cells expressing fusion proteins in the presence or absence of free donors and acceptors and obtained consistent results with other instruments and methods. This stable and low-cost spectral wide-field microscopic FRET imaging system provides a new toolbox for imaging molecular events with high spatial resolution in living cells.

One of the remaining challenges in functional connectivity (FC) studies is investigation of the temporal variability of FC networks. Recent studies focusing on the dynamic FC mostly use functional magnetic resonance imaging as an imaging tool to investigate the temporal variability of FC. We attempted to quantify this variability via analyzing the functional near-infrared spectroscopy (fNIRS) signals, which were recorded from the prefrontal cortex (PFC) of 12 healthy subjects during a Stroop test. Mutual information was used as a metric to determine functional connectivity between PFC regions. Two-dimensional correlation based similarity measure was used as a method to analyze within-subject and intersubject consistency of FC maps and how they change in time. We found that within-subject consistency (0.61±0.09) is higher than intersubject consistency (0.28±0.13). Within-subject consistency was not found to be task-specific. Results also revealed that there is a gradual change in FC patterns during a Stroop session for congruent and neutral conditions, where there is no such trend in the presence of an interference effect. In conclusion, we have demonstrated the between-subject, within-subject, and temporal variability of FC and the feasibility of using fNIRS for studying dynamic FC.

Fluorescence quantum yield (QY) indicates the efficiency of the fluorescence process. The QY of many fluorophores is sensitive to local tissue environments, highlighting the possibility of using QY as an indicator of important parameters such as pH or temperature. QY is commonly measured by comparison to a well-known standard in nonscattering media. We propose a new imaging method, called quantum yield imaging (QYI), to spatially map the QY of a fluorophore within anoptically diffusive media. QYI utilizes the wide-field diffuse optical technique spatial frequency domain imaging (SFDI) as well as planar fluorescence imaging. SFDI is used to measure the optical properties of the background media and the absorption contributed by the fluorophore. The unknown QY is then calculated by combining information from both modalities. A fluorescent sample with known QY is used to account for instrument response. To demonstrate QYI, rhodamine B and SNARF-5 were imaged in liquid phantoms with different background optical properties. The methanol:water ratio and pH were changed for rhodamine B and SNARF-5 solvents, respectively, altering the QY of each through a wide range.QYwasdeterminedwithanagreementof0.021and0.012forrhodamineBandSNARF-5,respectively.

We propose a saline injection-based method to quantify blood flow velocity in vivo with acoustic-resolution photoacoustic tomography. By monitoring the saline–blood interface propagating in the blood vessel, the flow velocity can be resolved. We first demonstrated our method in phantom experiments, where a root mean square error of prediction of 0.29  mm/s was achieved. By injecting saline into a mouse tail vein covered with 1 mm chicken tissue, we showed that the flow velocity in the tail vein could be measured at depths, which is especially pertinent to monitoring blood flow velocity in patients undergoing intravenous infusion.

Transcranial near-infrared (NIR) treatment of neurological diseases has gained recent momentum. However, the low NIR dose available to the brain, which shows severe scattering and absorption of the photons by human tissues, largely limits its effectiveness in clinical use. Hereby, we propose to take advantage of the strong scattering effect of the cranial tissues by applying an evenly distributed multiunit emitter array on the scalp to enhance the cerebral photon density while maintaining each single emitter operating under the safe thermal limit. By employing the Monte Carlo method, we simulated the transcranial propagation of the array emitted light and demonstrated markedly enhanced intracranial photon flux as well as improved uniformity of the photon distribution. These enhancements are correlated with the source location, density, and wavelength of light. To the best of our knowledge, we present the first systematic analysis of the intracranial light field established by the scalp-applied multisource array and reveal a strategy for the optimization of the therapeutic effects of the NIR radiation.

The development of tumor therapies based on the activation of antitumor immunity requires tumor models that are highly immunogenic. The immunologic response to fluorescent proteins, green fluorescent protein (GFP), or enhanced GFP (EGFP) was demonstrated in different cancer models. However, for live animal imaging, red and far-red fluorescent proteins are preferable, but their immunogenicity has not been studied. We assessed the immunogenicity of the red fluorescent protein, KillerRed (KR), in CT26 murine colon carcinoma. We showed a slower growth and a lower tumor incidence of KR-expressing tumors in comparison with nonexpressing ones. We found that KR-expressing lung metastases and rechallenged tumors were not formed in mice that had been surgically cured of KR-expressing primary tumors. The effect of low-dose cyclophosphamide (CY) treatment was also tested, as this is known to activate antitumor immune responses. The low-dose CY therapy of CT26-KR tumors resulted in inhibition of tumor growth and improved mouse survival. In summary, we have established a highly immunogenic tumor model that could be valuable for investigations of the mechanisms of antitumor immunity and the development of new therapeutic approaches.

A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N-hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg, respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization.

The discovery that a pulsed laser could trigger an auditory neural response inspired ongoing research on cochlear implants activated by optical stimulus rather than by electrical current. However, most studies to date have used visible light (532 nm) or long-wavelength near-infrared (<1840  nm) and involved making a hole in the cochlea. This paper investigates the effect of optical parameters on the optically evoked compound action potentials (oCAPs) from the guinea pig cochlea, using a pulsed semiconductor near-infrared laser (980 nm) without making a hole in the cochlea. Synchronous trigger laser pulses were used to stimulate the cochlea, before and after deafening, upon varying the pulse duration (30–1000  μs) and an amount of radiant energy (0–53.2  mJ/cm²). oCAPs were successfully recorded after deafening. The amplitude of the oCAPs increased as the infrared radiant energy was increased at a fixed 50  μs pulse duration, and decreased with a longer pulse duration at a fixed 37.1  mJ/cm² radiant energy. The latency of the oCAPs shortened with increasing radiant energy at a fixed pulse duration. With a higher stimulation rate, the amplitude of the oCAPs’ amplitude decreased.