Optical property recovery with spatially-resolved diffuse reflectance at short source-detector separations using a compact fiber-optic probe

We describe a compact fiber-optic probe (2 mm outside diameter) that utilizes spatially-resolved diffuse reflectance for tissue optical property recovery. Validation was performed in phantoms containing Intralipid 20% as scatterer, and methylene blue (MB), MnTPPS, and/or India ink as absorbers. Over a range of conditions, the reduced scattering coefficient was recovered with a root mean square error (RMSE) of 0.86-2.7 cm^-1 (average error = 3.8%). MB concentration was recovered with RMSE = 0.26-0.52 µM (average error = 15.0%), which did not vary with inclusion of MnTPPS (p=0.65). This system will be utilized to determine optical properties in human abscesses, in order to generate treatment plans for photodynamic therapy.     

Combined autofluorescence and diffuse reflectance for brain tumour surgical guidance: initial ex vivo study results

This ex vivo study was conducted to assess the potential of using a fibre optic probe system based on autofluorescence and diffuse reflectance for tissue differentiation in the brain. A total of 180 optical measurements were acquired from 28 brain specimens (five patients) with eight excitation and emission wavelengths spanning from 300 to 700 nm. Partial least square-linear discriminant analysis (PLS-LDA) was used for tissue discrimination. Leave-one-out cross validation (LOOCV) was then used to evaluate the performance of the classification model. Grey matter was differentiated from tumour tissue with sensitivity of 89.3% and specificity of 92.5%. The variable importance in projection (VIP) derived from the PLS regression was applied to wavelengths selection, and identified the biochemical sources of the detected signals. The initial results of the study were promising and point the way towards a cost-effective, miniaturized hand-held probe for real time and label-free surgical guidance.     

Improving the depth sensitivity of time-resolved measurements by extracting the distribution of times-of-flight

Time-resolved (TR) techniques provide a means of discriminating photons based on their time-of-flight. Since early arriving photons have a lower probability of probing deeper tissue than photons with long time-of-flight, time-windowing has been suggested as a method for improving depth sensitivity. However, TR measurements also contain instrument contributions (instrument-response-function, IRF), which cause temporal broadening of the measured temporal point-spread function (TPSF) compared to the true distribution of times-of-flight (DTOF). The purpose of this study was to investigate the influence of the IRF on the depth sensitivity of TR measurements. TPSFs were acquired on homogeneous and two-layer tissue-mimicking phantoms with varying optical properties. The measured IRF and TPSFs were deconvolved using a stable algorithm to recover the DTOFs. The microscopic Beer-Lambert law was applied to the TPSFs and DTOFs to obtain depth-resolved absorption changes. In contrast to the DTOF, the latest part of the TPSF was not the most sensitive to absorption changes in the lower layer, which was confirmed by computer simulations. The improved depth sensitivity of the DTOF was illustrated in a pig model of the adult human head. Specifically, it was shown that dynamic absorption changes obtained from the late part of the DTOFs recovered from TPSFs acquired by probes positioned on the scalp were similar to absorption changes measured directly on the brain. These results collectively demonstrate that this method improves the depth sensitivity of TR measurements by removing the effects of the IRF.OCIS codes: (170.3660) Light propagation in tissues, (170.3890) Medical optics instrumentation     

Determination of reference values for optical properties of liquid phantoms based on Intralipid and India ink

A multi-center study has been set up to accurately characterize the optical properties of diffusive liquid phantoms based on Intralipid and India ink at near-infrared (NIR) wavelengths. Nine research laboratories from six countries adopting different measurement techniques, instrumental set-ups, and data analysis methods determined at their best the optical properties and relative uncertainties of diffusive dilutions prepared with common samples of the two compounds. By exploiting a suitable statistical model, comprehensive reference values at three NIR wavelengths for the intrinsic absorption coefficient of India ink and the intrinsic reduced scattering coefficient of Intralipid-20% were determined with an uncertainty of about 2% or better, depending on the wavelength considered, and 1%, respectively. Even if in this study we focused on particular batches of India ink and Intralipid, the reference values determined here represent a solid and useful starting point for preparing diffusive liquid phantoms with accurately defined optical properties. Furthermore, due to the ready availability, low cost, long-term stability and batch-to-batch reproducibility of these compounds, they provide a unique fundamental tool for the calibration and performance assessment of diffuse optical spectroscopy instrumentation intended to be used in laboratory or clinical environment. Finally, the collaborative work presented here demonstrates that the accuracy level attained in this work for optical properties of diffusive phantoms is reliable.OCIS codes: (170.5280) Photon migration, (170.7050) Turbid media, (170.3890) Medical optics instrumentation     

Frequency-modulated light scattering interferometry employed for optical properties and dynamics studies of turbid media

In the present work, fiber-based frequency-modulated light scattering interferometry (FMLSI) is developed and employed for studies of optical properties and dynamics in liquid phantoms made from Intralipid^®. The fiber-based FMLSI system retrieves the optical properties by examining the intensity fluctuations through the turbid medium in a heterodyne detection scheme using a continuous-wave frequency-modulated coherent light source. A time resolution of 21 ps is obtained, and the experimental results for the diluted Intralipid phantoms show good agreement with the predicted results based on published data. The present system shows great potential for assessment of optical properties as well as dynamic studies in liquid phantoms, dairy products, and human tissues.OCIS codes: (290.7050) Turbid media, (120.5820) Scattering measurements, (120.6160) Speckle interferometry     

Measurements of extrinsic fluorescence in Intralipid and polystyrene microspheres

The fluorescence of Intralipid and polystyrene microspheres with sphere diameter of 1 µm at a representative lipid and microsphere concentration for simulation of mucosal tissue scattering has not been a subject of extensive experimental study. In order to elucidate the quantitative relationship between lipid and microsphere concentration and the respective fluorescent intensity, the extrinsic fluorescence spectra between 360 nm and 650 nm (step size of 5 nm) were measured at different lipid concentrations (from 0.25% to 5%) and different microsphere concentrations (0.00364, 0.0073, 0.0131 spheres per cubic micrometer) using laser excitation at 355 nm with pulse energy of 2.8 µJ. Current findings indicated that Intralipid has a broadband emission between 360 and 650 nm with a primary peak at 500 nm and a secondary peak at 450 nm while polystyrene microspheres have a single peak at 500 nm. In addition, for similar scattering properties the fluorescence of Intralipid solutions is approximately three-fold stronger than that of the microsphere solutions. Furthermore, Intralipid phantoms with lipid concentrations ~2% (simulating the bottom layer of mucosa) produce up to seven times stronger fluorescent emission than phantoms with lipid concentration ~0.25% (simulating the top layer of mucosa). The fluoresence decays of Intralipid and microsphere solutions were also recorded for estimation of fluorescence lifetime.OCIS codes: (170.3650) Lifetime-based sensing, (170.6280) Spectroscopy, fluorescence and luminescence     

Ultrahigh resolution spectral-domain optical coherence tomography using the 1000–1600 nm spectral band

Ultrahigh resolution optical coherence tomography (UHR-OCT) can image microscopic features that are not visible with the standard OCT resolution of 5-15 µm. In previous studies, high-speed UHR-OCT has been accomplished within the visible (VIS) and near-infrared (NIR-I) spectral ranges, specifically within 550-950 nm. Here, we present a spectral domain UHR-OCT system operating in a short-wavelength infrared (SWIR) range from 1000 to 1600 nm using a supercontinuum light source and an InGaAs-based spectrometer. We obtained an axial resolution of 2.6 µm in air, the highest ever recorded in the SWIR window to our knowledge, with deeper penetration into tissues than VIS or NIR-I light. We demonstrate imaging of conduction fibers of the left bundle branch in freshly excised porcine hearts. These results suggest a potential for deep-penetration, ultrahigh resolution OCT in intraoperative applications.     

Single-laser-based simultaneous four-wavelength excitation source for femtosecond two-photon fluorescence microscopy

Multicolor labeling of biological samples with large volume is required for omic-level of study such as the construction of nervous system connectome. Among the various imaging method, two photon microscope has multiple advantages over traditional single photon microscope for higher resolution and could image large 3D volumes of tissue samples with superior imaging depth. However, the growing number of fluorophores for labeling underlines the urgent need for an ultrafast laser source with the capability of providing simultaneous plural excitation wavelengths for multiple fluorophores. Here, we propose and demonstrate a single-laser-based four-wavelength excitation source for two-photon fluorescence microscopy. Using a sub-100 fs 1,070-nm Yb:fiber laser to pump an ultrashort nonlinear photonic crystal fiber in the low negative dispersion region, we introduced efficient self-phase modulation and acquired a blue-shifted spectrum dual-peaked at 812 and 960 nm with 28.5% wavelength conversion efficiency. By compressing the blue-shift near-IR spectrum to 33 fs to ensure the temporal overlap of the 812 and 960 nm peaks, the so-called sum frequency effect created the third virtual excitation wavelength effectively at 886 nm. Combined with the 1,070 nm laser source as the fourth excitation wavelength, the all-fiber-format four-wavelength excitation source enabled simultaneous four-color two-photon imaging in Brainbow AAV-labeled (TagBFP, mTFP, EYFP, and mCherry) brain samples. With an increased number of excitation wavelengths and improved excitation efficiency than typical commercial femtosecond lasers, our compact four-wavelength excitation approach can provide a versatile, efficient, and easily accessible solution for multiple-color two-photon fluorescence imaging in the field of neuroscience, biomolecular probing, and clinical applications with at least four spectrally-distinct fluorophores.     

Diamond Raman laser and Yb fiber amplifier for in vivo multiphoton fluorescence microscopy

Here we introduce a fiber amplifier and a diamond Raman laser that output high powers (6.5 W, 1.3 W) at valuable wavelengths (1060 nm, 1250 nm) for two-photon excitation of red-shifted fluorophores. These custom excitation sources are both simple to construct and cost-efficient in comparison to similar custom and commercial alternatives. Furthermore, they operate at a repetition rate (80 MHz) that allows fast image acquisition using resonant scanners. With our system we demonstrate compatibility with fast resonant scanning, the ability to acquire neuronal images, and the capability to image vasculature at deep locations (>1 mm) within the mouse cerebral cortex.     

Blue-LIRIC in the rabbit cornea: efficacy,tissue effects,and repetition rate scaling

Laser-induced refractive index change (LIRIC) is being developed as a non-invasive way to alter optical properties of transparent, ophthalmic materials including corneas ex vivo and in vivo. This study examined the optical and biological effects of blue-LIRIC (wavelengths 400–405 nm) of ex-vivo rabbit corneas. Following LIRIC treatment at low and high repetition rates (8.3 MHz and 80 MHz, respectively), we interferometrically measured optical phase change, obtained transmission electron microscopy (TEM) micrographs, and stained histological sections with collagen hybridizing peptides (CHP) to assess the structural and organizational changes caused by LIRIC at different repetition rates. Finally, we performed power and scan speed scaling experiments at three different repetition rates (1 MHz, 8.3 MHz, and 80 MHz) to study their impact on LIRIC efficacy. Histologic co-localization of CHP and LIRIC-generated green autofluorescence signals suggested that collagen denaturation had occurred in the laser-irradiated region. TEM imaging showed different ultrastructural modifications for low and high repetition rate writing, with discrete homogenization of collagen fibrils at 80 MHz, as opposed to contiguous homogenization at 8.3 MHz. Overall, this study confirmed that LIRIC efficacy can be dramatically increased, while still avoiding tissue ablation, by lowering the repetition rate from 80 MHz to 8.3 MHz. Modeling suggests that this is due to a higher, single-pulse, energy density deposition at given laser powers during 8.3 MHz LIRIC.     

Two-dimensional (2D) dynamic vibration optical coherence elastography (DV-OCE) for evaluating mechanical properties: a potential application in tissue engineering

Mechanical properties in tissues are an important indicator because they are associated with disease states. One of the well-known excitation sources in optical coherence elastography (OCE) to determine mechanical properties is acoustic radiation force (ARF); however, a complicated focusing alignment cannot be avoided. Another excitation source is a piezoelectric (PZT) stack to obtain strain images via compression, which can affect the intrinsic mechanical properties of tissues in tissue engineering. In this study, we report a new technique called two-dimensional (2D) dynamic vibration OCE (DV-OCE) to evaluate 2D wave velocities without tedious focusing alignment procedures and is a non-contact method with respect to the samples. The three-dimensional (3D) Fourier transform was utilized to transfer the traveling waves (x, y, t) into 3D k-space (k_x, k_y, f). A spatial 2D wavenumber filter and multi-angle directional filter were employed to decompose the waves with omni-directional components into four individual traveling directions. The 2D local wave velocity algorithm was used to calculate a 2D wave velocity map. Six materials, two homogeneous phantoms with 10 mm thickness, two homogeneous phantoms with 2 mm thickness, one heterogeneous phantom with 2 mm diameter inclusion and an ex vivo porcine kidney, were examined in this study. In addition, the ARF-OCE was used to evaluate wave velocities for comparison. Numerical simulations were performed to validate the proposed 2D dynamic vibration OCE technique. We demonstrate that the experimental results were in a good agreement with the results from ARF-OCE (transient OCE) and numerical simulations. Our proposed 2D dynamic vibration OCE could potentially pave the way for mechanical evaluation in tissue engineering and for laboratory translation with easy-to-setup and contactless advantages.     

Narrowband diffuse reflectance spectroscopy in the 900–1000 nm wavelength region to quantify water and lipid content of turbid media

We report a narrow wavelength band diffuse reflectance spectroscopy (nb-DRS) method to determine water and fat ratios of scattering media in the 900–1000 nm range. This method was shown to be linearly correlated with absolute water and fat concentrations as tested on a set of turbid emulsion phantoms with a range of water and lipid compositions. Robustness to scattering assumptions was demonstrated and compared against measured scattering by a frequency-domain photon migration system. nb-DRS was also tested on ex-vivo porcine samples and compared against direct tissue water extraction by analytical chemistry methods. We speculate nb-DRS has potential applications in portable devices such as clinical and digital health wearables.     

Compressed sensing time-resolved spectrometer for quantification of light absorbers in turbid media

Time-resolved (TR) spectroscopy is well-suited to address the challenges of quantifying light absorbers in highly scattering media such as living tissue; however, current TR spectrometers are either based on expensive array detectors or rely on wavelength scanning. Here, we introduce a TR spectrometer architecture based on compressed sensing (CS) and time-correlated single-photon counting. Using both CS and basis scanning, we demonstrate that—in homogeneous and two-layer tissue-mimicking phantoms made of Intralipid and Indocyanine Green—the CS method agrees with or outperforms uncompressed approaches. Further, we illustrate the superior depth sensitivity of TR spectroscopy and highlight the potential of the device to quantify absorption changes in deeper (>1 cm) tissue layers.     

Investigation of the scattering and attenuation properties of cataracts formed in mouse eyes with 1060-nm and 1310-nm swept-source optical coherence tomography

Cataracts are the leading cause of blindness worldwide. Here we propose optical coherence tomography (OCT) as a quantitative method for investigating cataracts. OCT provides volumetric and non-invasive access to the lens and makes it possible to rapidly observe the formation of opacifications in animal models such as mice. We compared the performance of two different wavelengths – 1060 nm and 1310 nm – for OCT imaging in cataract research. In addition, we present multi-contrast OCT capable of mapping depth-resolved scattering and average anterior cortical attenuation properties of the crystalline lens and quantitatively characterize induced cataract development in the mouse eye. Lastly, we also propose a novel method based on the retinal OCT projection image for quantifying and mapping opacifications in the lens, which showed a good correlation with scattering and attenuation characteristics simultaneously analyzed during the process of cataract formation in the lens.     

Optimization of photo-biomodulation therapy for wound healing of diabetic foot ulcers in vitro and in vivo

Unclear optical parameters make photo-biomodulation (PBM) difficult to implement in diabetic foot ulcer (DFU) clinically. Here, 12 wavelengths (400–900 nm) were used to conduct PBM to heal DFU wounds in vitro and in vivo. PBM at 10 mW/cm² and 0.5–4 J/cm² with all 12 wavelengths promoted proliferation of diabetic wound cells. In a mimic DFU (mDFU) rat model, PBM (425, 630, 730, and 850 nm, and a combination light strategy) promoted mDFU healing. The positive cell proliferation, re-epithelialization, angiogenesis, collagen synthesis, and inflammation were possible mechanisms. The combination strategy had the best effect, which can be applied clinically.     

Do different turbid media with matched bulk optical properties also exhibit similar polarization properties?

We here investigate polarimetric behavior of thick samples of porcine liver, Intralipid, and microsphere-based tissue phantoms whose absorption and scattering properties are matched. Using polarized light we measured reflection mode Mueller matrices and derived linear/circular/total depolarization rates, based on polar decomposition. According to our results, phantoms exhibit greater depolarization rates in the backscattering geometry than the liver sample. The enhanced tissue polarization preservation differs from previous reports of polarimetric transmission studies, with the likely cause of this difference being the angular dependence of the single-scattering phase function. Also, Intralipid approximated polarimetric liver behavior well, whereas the polystyrene phantoms did not.     

First-in-clinical application of a time-gated diffuse correlation spectroscopy system at 1064 nm using superconducting nanowire single photon detectors in a neuro intensive care unit

Recently proposed time-gated diffuse correlation spectroscopy (TG-DCS) has significant advantages compared to conventional continuous wave (CW)-DCS, but it is still in an early stage and clinical capability has yet to be established. The main challenge for TG-DCS is the lower signal-to-noise ratio (SNR) when gating for the deeper traveling late photons. Longer wavelengths, such as 1064 nm have a smaller effective attenuation coefficient and a higher power threshold in humans, which significantly increases the SNR. Here, we demonstrate the clinical utility of TG-DCS at 1064 nm in a case study on a patient with severe traumatic brain injury admitted to the neuro-intensive care unit (neuroICU). We showed a significant correlation between TG-DCS early (ρ = 0.67) and late (ρ = 0.76) gated against invasive thermal diffusion flowmetry. We also analyzed TG-DCS at high temporal resolution (50 Hz) to elucidate pulsatile flow data. Overall, this study demonstrates the first clinical translation capability of the TG-DCS system at 1064 nm using a superconducting nanowire single-photon detector.     

Performance improvement of a silicon nitride ring resonator biosensor operated in the TM mode at 1310 nm

Silicon-based ring resonators have been demonstrated to be a key element to build lab-on-chip devices due to their ability to perform as label-free photonic sensors. In this work, we demonstrate photonic biosensing using silicon nitride ring resonators operated in the TM mode around 1310 nm wavelengths. Our results show that operating the devices using the TM mode results in an increased sensitivity in comparison with the typically used TE mode, while working at 1310 nm wavelengths compared to 1550 nm contributes to an increased quality factor. As a result, a reduction in the intrinsic limit of detection is achieved, indicating the suitability of TM modes in the 1310 nm regime for biosensing using integrated photonics.     

Phase-sensitive OCT imaging of multiple nanoparticle species using spectrally multiplexed single pulse photothermal excitation

We apply phase-sensitive optical coherence tomography to image multiple nanoparticle species with two excitation wavelengths matched to their distinct absorption peaks. Using different modulation frequencies, multiple species collocated within the sample can be distinguished. In addition, we characterize single-pulse excitation schemes as a method to minimize bulk heating of the sample. We demonstrate this new scheme with B-mode photothermal measurements of tissue phantoms.OCIS codes: (170.4500) Optical coherence tomography, (160.4236) Nanomaterials, (110.3000) Image quality assessment     

Effect of dependent scattering on the optical properties of Intralipid tissue phantoms

The calibration of optical tissue-simulating phantoms remains an open question in spite of the many techniques proposed for accurate measurements of optical properties. As a consequence, a reference phantom with well known optical properties is still missing. As a first step towards a reference phantom we have recently proposed to use dilutions of Intralipid 20%. In this paper we discuss a matter that is commonly ignored when dilutions are prepared, i.e., the possibility of deviations from the simple linear relationships between the optical properties of the dilution and the Intralipid concentration due to the effects of dependent scattering. The results of an experimental investigation showed that dependent scattering does not affect absorption. As for the reduced scattering coefficient the effect can be described adding a term proportional to the square of the concentration. However, for concentrations of interest for tissue optics deviations from linearity remain within about 2%. The experimental investigation also showed that the microphysical properties of Intralipid are not affected by dilution. These results show the possibility to easily obtain a liquid diffusive phantom whose optical properties are known with error smaller than about 1%. Due to the intrinsic limitations of the different techniques proposed for measuring the optical properties it seems difficult to obtain a similar accuracy for solid phantoms.