Rare-earth-doped nanoparticles are one of the emerging probes for bioimaging due to their visible-to-near-infrared (NIR) upconversion emission via sequential single-photon absorption at NIR wavelengths. The NIR-excited upconversion property and high photostability make this probe appealing for deep tissue imaging. So far, upconversion nanoparticles include ytterbium ions (Yb^3  +) codoped with other rare earth ions, such as erbium (Er^3  +) and thulium (Tm^3  +). In these types of upconversion nanoparticles, through energy transfer from Yb^3  + excited with continuous wave light at a wavelength of 980 nm, upconversion emission of the other rare earth dopants is induced. We have found that the use of the excitation of Er^3  + in the 1550-nm wavelength region allows us to perform deep tissue imaging with reduced degradation of spatial resolution. In this excitation–emission process, three and four photons of 1550-nm light are sequentially absorbed, and Er^3  + emits photons in the 550- and 660-nm wavelength regions. We demonstrate that, compared with the case using 980-nm wavelength excitation, the use of 1550-nm light enables us to moderate degradation of spatial resolution in deep tissue imaging due to the lower light scattering coefficient compared with 980-nm light. We also demonstrate that live cell imaging is feasible with this 1550 nm excitation.

We present three-dimensional (3-D) high-resolution spectral-domain optical coherence microscopy (SD-OCM) by using a supercontinuum (SC) fiber laser source with 300-nm spectral bandwidth (full-width at half-maximum) in the 1700-nm spectral band. By using low-coherence interferometry with SC light and a confocal detection scheme, we realized lateral and axial resolutions of 3.4 and 3.8  μm in tissue (n  =  1.38), respectively. This is, to the best of our knowledge, the highest 3-D spatial resolution reported among those of Fourier-domain optical coherence imaging techniques in the 1700-nm spectral band. In our SD-OCM, to enhance the imaging depth, a full-range method was implemented, which suppressed the formation of a coherent ghost image and allowed us to set the zero-delay position inside the samples. We demonstrated the 3-D high-resolution imaging capability of 1700-nm SD-OCM through the measurement of an interference signal from a mirror surface and imaging of a single 200-nm polystyrene bead and a pig thyroid gland. Deep tissue imaging at a depth of up to 1.8 mm was also demonstrated. This is the first demonstration of 3-D high-resolution SD-OCM in the 1700-nm spectral band.

To refine animal research, vital signs, activity, stress, and pain must be monitored. In chronic studies, some measures can be assessed using telemetry sensors. Although this methodology provides high-precision data, an initial surgery for device implantation is necessary, potentially leading to stress, wound infections, and restriction of motion. Recently, camera systems have been adapted for animal research. We give an overview of parameters that can be assessed using imaging in the visible, near-infrared, and thermal spectrum of light. It focuses on heart activity, respiration, oxygen saturation, and motion, as well as on wound analysis. For each parameter, we offer recommendations on the minimum technical requirements of appropriate systems, regions of interest, and light conditions, among others. In general, these systems demonstrate great performance. For heart and respiratory rate, the error was <4  beats  /  min and 5 breaths/min. Furthermore, the systems are capable of tracking animals during different behavioral tasks. Finally, studies indicate that inhomogeneous temperature distribution around wounds might be an indicator of (pending) infections. In sum, camera-based techniques have several applications in animal research. As vital parameters are currently only assessed in sedated animals, the next step should be the integration of these modalities in home-cage monitoring.

This guest editorial introduces the Special Section on Spatial Frequency Domain Imaging.

Diffuse optical imaging probes deep living tissue enabling structural, functional, metabolic, and molecular imaging. Recently, due to the availability of spatial light modulators, wide-field quantitative diffuse optical techniques have been implemented, which benefit greatly from structured light methodologies. Such implementations facilitate the quantification and characterization of depth-resolved optical and physiological properties of thick and deep tissue at fast acquisition speeds. We summarize the current state of work and applications in the three main techniques leveraging structured light: spatial frequency-domain imaging, optical tomography, and single-pixel imaging. The theory, measurement, and analysis of spatial frequency-domain imaging are described. Then, advanced theories, processing, and imaging systems are summarized. Preclinical and clinical applications on physiological measurements for guidance and diagnosis are summarized. General theory and method development of tomographic approaches as well as applications including fluorescence molecular tomography are introduced. Lastly, recent developments of single-pixel imaging methodologies and applications are reviewed.

We present a Monte Carlo (MC) method to determine depth-dependent probability distributions of photon visitation and detection for optical reflectance measurements performed in the spatial frequency domain (SFD). These distributions are formed using an MC simulation for radiative transport that utilizes a photon packet weighting procedure consistent with the two-dimensional spatial Fourier transform of the radiative transport equation. This method enables the development of quantitative metrics for SFD optical sampling depth in layered tissue and its dependence on both tissue optical properties and spatial frequency. We validate the computed depth-dependent probability distributions using SFD measurements in a layered phantom system with a highly scattering top layer of variable thickness supported by a highly absorbing base layer. We utilize our method to establish the spatial frequency-dependent optical sampling depth for a number of tissue types and also provide a general tool to determine such depths for tissues of arbitrary optical properties.

A method to correct for surface scattering in spatial frequency domain imaging (SFDI) is presented. The use of a modified analytical solution of the radiative transfer equation allows calculation of the reflectance and the phase of a rough semi-infinite geometry so that both spatial frequency domain reflectance and phase can be applied for precise retrieval of the bulk optical properties and the surface scattering. For validation of the method, phantoms with different surface roughness were produced. Contrarily, with the modified theory, it was possible to dramatically reduce systematic errors due to surface scattering. The evaluation of these measurements with the state-of-the-art theory and measuring modality, i.e., using crossed linear polarizers, reveals large errors in the determined optical properties, depending on the surface roughness, of up to ≈100  %  . These results were confirmed with SFDI measurements on a phantom that has a structured rough surface.

This study aims to determine if light scatter parameters measured with spatial frequency domain imaging (SFDI) can accurately predict stromal, epithelial, and adipose fractions in freshly resected, unstained human breast specimens. An explicit model was developed to predict stromal, epithelial, and adipose fractions as a function of light scattering parameters, which was validated against a quantitative analysis of digitized histology slides for N  =  31 specimens using leave-one-out cross-fold validation. Specimen mean stromal, epithelial, and adipose volume fractions predicted from light scattering parameters strongly correlated with those calculated from digitized histology slides (r  =  0.90, 0.77, and 0.91, respectively, p-value <1  ×  10  −  6). Additionally, the ratio of predicted epithelium to stroma classified malignant specimens with a sensitivity and specificity of 90% and 81%, respectively, and also classified all pixels in malignant lesions with 63% and 79%, at a threshold of 1. All specimens and pixels were classified as malignant, benign, or fat with 84% and 75% accuracy, respectively. These findings demonstrate how light scattering parameters acquired with SFDI can be used to accurately predict and spatially map stromal, epithelial, and adipose proportions in fresh unstained, human breast tissue, and suggest that these estimations could provide diagnostic value.

Fast estimation of optical properties from reflectance measurements at two spatial frequencies could pave way for real-time, wide-field and quantitative mapping of vital signs of tissues. We present a machine learning-based approach for estimating optical properties in the spatial frequency domain, where a random forest regression algorithm is trained over data obtained from Monte-Carlo photon transport simulations. The algorithm learns the nonlinear mapping between diffuse reflectance at two spatial frequencies, and the absorption and reduced scattering coefficient of the tissue under consideration. Using this method, absorption and reduced scattering properties could be obtained over a 1 megapixel image in 450 ms with errors as low as 0.556% in absorption and 0.126% in reduced scattering.

We present a method to recover absorption and reduced scattering spectra for each layer of a two-layer turbid media from spatial frequency-domain spectroscopy data. We focus on systems in which the thickness of the top layer is less than the transport mean free path   (  0.1  −  0.8l^*  )  . We utilize an analytic forward solver, based upon the N’th-order spherical harmonic expansion with Fourier decomposition   (  SHEF_N  )   method in conjunction with a multistage inverse solver. We test our method with data obtained using spatial frequency-domain spectroscopy with 32 evenly spaced wavelengths within λ  =  450 to 1000 nm on six-layered tissue phantoms with distinct optical properties. We demonstrate that this approach can recover absorption and reduced scattering coefficient spectra for both layers with accuracy comparable with current Monte Carlo methods but with lower computational cost and potential flexibility to easily handle variations in parameters such as the scattering phase function or material refractive index. To our knowledge, this approach utilizes the most accurate deterministic forward solver used in such problems and can successfully recover properties from a two-layer media with superficial layer thicknesses.

Mapping the optical absorption and scattering properties of tissues using spatial frequency-domain imaging (SFDI) enhances quantitative fluorescence imaging of protoporphyrin IX (PpIX) in gliomas in the preclinical setting. The feasibility of using SFDI in the operating room was investigated here. A benchtop SFDI system was modified to mount directly to a commercial operating microscope. A digital light processing module imposed a selectable spatial light pattern from a broad-band xenon arc lamp to illuminate the surgical field. White light excitation and a liquid crystal-tunable filter allowed the diffuse reflectance images to be recorded at discrete wavelengths from 450 to 720 nm on a sCMOS camera. The performance was first tested in tissue-simulating phantoms, and data were then acquired intraoperatively during brain tumor resection surgery. The optical absorption and transport scattering coefficients could be estimated with average errors of 3.2% and 4.5% for the benchtop and clinical systems, respectively, with spatial resolution of better than 0.7 mm. These findings suggest that SFDI can be implemented in a clinically relevant configuration to achieve accurate mapping of the optical properties in the surgical field that can then be applied to achieve quantitative imaging of the fluorophore.

Imaging Cherenkov emission during radiotherapy permits real-time visualization of external beam delivery on superficial tissue. This signal is linear with absorbed dose in homogeneous media, indicating potential for quantitative dosimetry. In humans, the inherent heterogeneity of tissue optical properties (primarily from blood and skin pigment) distorts the linearity between detected Cherenkov signal and absorbed dose. We examine the potential to correct for superficial vasculature using spatial frequency domain imaging (SFDI) to map tissue optical properties for large fields of view. In phantoms, applying intensity corrections to simulate blood vessels improves Cherenkov image (CI) negative contrast by 24% for a vessel 1.9-mm-in diameter. In human trials, SFDI and CI are acquired for women undergoing whole breast radiotherapy. Applied corrections reduce heterogeneity due to vasculature within the sampling limits of the SFDI from a 22% difference as compared to the treatment plan, down to 6% in one region and from 14% down to 4% in another region. The optimal use for this combined imaging system approach is to correct for small heterogeneities such as superficial blood vessels or for interpatient variations in blood/melanin content such that the corrected CI more closely represents the surface dose delivered.

Quantitative diffuse optical imaging has the potential to provide valuable functional information about tissue status, such as oxygen saturation or blood content to healthcare practitioners in real time. However, significant technical challenges have so far prevented such tools from being deployed in the clinic. Toward achieving this goal, prior research introduced methods based on spatial frequency domain imaging (SFDI) that allow real-time (within milliseconds) wide-field imaging of optical properties but at a single wavelength. However, for this technology to be useful to clinicians, images must be displayed in terms of metrics related to the physiological state of the tissue, hence interpretable to guide decision-making. For this purpose, recent developments introduced multispectral SFDI methods for rapid imaging of oxygenation parameters up to 16 frames per seconds (fps). We introduce real-time, wide-field, and quantitative blood parameters imaging using spatiotemporal modulation of light. Using this method, we are able to quantitatively obtain optical properties at 100 fps at two wavelengths (665 and 860 nm), and therefore oxygenation, oxyhemoglobin, and deoxyhemoglobin, using a single camera with, at most, 4.2% error in comparison with standard SFDI acquisitions.

Imaging methods permitting real-time, wide-field, and quantitative optical mapping of biological tissue properties offer an unprecedented range of applications for clinical use. Following the development of spatial frequency domain imaging, we introduce a real-time demodulation method called single snapshot of optical properties (SSOPs). However, since this method uses only a single image to generate absorption and reduced scattering maps, it was limited by a degraded image quality resulting in artifacts that diminished its potential for clinical use. We present filtering strategies for improving the image quality of optical properties maps obtained using SSOPs. We investigate the effect of anisotropic two-dimensional filtering strategies for spatial frequencies ranging from 0.1 to 0.4  mm^−  1 directly onto N  =  10 hands. Both accuracy and image quality of the optical properties are quantified in comparison with standard, multiple image acquisitions in the spatial frequency domain. Overall, using optimized filters, mean errors in predicting optical properties using SSOP remain under 8.8% in absorption and 7.5% in reduced scattering, while significantly improving image quality. Overall this work contributes to advance real-time, wide-field, and quantitative diffuse optical imaging toward clinical evaluation.

We present the effects of using a single-pixel camera approach to extract optical properties with the single-snapshot spatial frequency-domain imaging method. We acquired images of a human hand for spatial frequencies ranging from 0.1 to 0.4  mm^−  1 with increasing compression ratios using adaptive basis scan wavelet prediction strategy. In summary, our findings indicate that the extracted optical properties remained usable up to 99% of compression rate at a spatial frequency of 0.2  mm^−  1 with errors of 5% in reduced scattering and 10% in absorption.

Spatial frequency domain imaging (SFDI) has witnessed very rapid growth over the last decade, owing to its unique capabilities for imaging optical properties and chromophores over a large field-of-view and in a rapid manner. We provide a comprehensive review of the principles of this imaging method as of 2019, review the modeling of light propagation in this domain, describe acquisition methods, provide an understanding of the various implementations and their practical limitations, and finally review applications that have been published in the literature. Importantly, we also introduce a group effort by several key actors in the field for the dissemination of SFDI, including publications, advice in hardware and implementations, and processing code, all freely available online.

We introduce a method for quantitative hyperspectral optical imaging in the spatial frequency domain (hs-SFDI) to image tissue absorption (μ_a) and reduced scattering ( μs′) parameters over a broad spectral range. The hs-SFDI utilizes principles of spatial scanning of the spectrally dispersed output of a supercontinuum laser that is sinusoidally projected onto the tissue using a digital micromirror device. A scientific complementary metal–oxide–semiconductor camera is used for capturing images that are demodulated and analyzed using SFDI computational models. The hs-SFDI performance is validated using tissue-simulating phantoms over a range of μ_a and μs′ values. Quantitative hs-SFDI images are obtained from an ex-vivo beef sample to spatially resolve concentrations of oxy-, deoxy-, and met-hemoglobin, as well as water and fat fractions. Our results demonstrate that the hs-SFDI can quantitatively image tissue optical properties with 1000 spectral bins in the 580- to 950-nm range over a wide, scalable field of view. With an average accuracy of 6.7% and 12.3% in μ_a and μs′, respectively, compared to conventional methods, hs-SFDI offers a promising approach for quantitative hyperspectral tissue optical imaging.

There is a need for noninvasive, quantitative methods to characterize wound healing in the context of longitudinal investigations related to regenerative medicine. Such tools have the potential to inform the assessment of wound status and healing progression and aid the development of new treatments. We employed spatial frequency domain imaging (SFDI) to characterize the changes in optical properties of tissue during wound healing progression in a porcine model of split-thickness skin grafts and also in a model of burn wound healing with no graft intervention. Changes in the reduced scattering coefficient measured using SFDI correlated with structural changes reported by histology of biopsies taken concurrently. SFDI was able to measure spatial inhomogeneity in the wounds and predicted heterogeneous healing. In addition, we were able to visualize differences in healing rate, depending on whether a wound was debrided and grafted, versus not debrided and left to heal without intervention apart from topical burn wound care. Changes in the concentration of oxy- and deoxyhemoglobin were also quantified, giving insight into hemodynamic changes during healing.

Previous work has shown that capturing optical emission from plastic discs attached directly to the skin can be a viable means to accurately measure surface dose during total skin electron therapy. This method can provide accurate dosimetric information rapidly and remotely without the need for postprocessing. The objective of this study was to: (1) improve the robustness and usability of the scintillators and (2) enhance sensitivity of the optical imaging system to improve scintillator emission detection as related to tissue surface dose. Baseline measurements of scintillator optical output were obtained by attaching the plastic discs to a flat tissue phantom and simultaneously irradiating and imaging them. Impact on underlying surface dose was evaluated by placing the discs on-top of the active element of an ionization chamber. A protective coating and adhesive backing were added to allow easier logistical use, and they were also subjected to disinfection procedures, while verifying that these changes did not affect the linearity of response with dose. The camera was modified such that the peak of detector quantum efficiency better overlapped with the emission spectra of the scintillating discs. Patient imaging was carried out and surface dose measurements were captured by the updated camera and compared to those produced by optically stimulated luminescence detectors (OSLD). The updated camera was able to measure surface dose with <3  %   difference compared to OSLD–Cherenkov emission from the patient was suppressed and scintillation detection was enhanced by 25  ×   and 7  ×  , respectively. Improved scintillators increase underlying surface dose on average by 5.2  ±  0.1  %   and light output decreased by 2.6  ±  0.3  %  . Disinfection had <0.02  %   change on scintillator light output. The enhanced sensitivity of the imaging system to scintillator optical emission spectrum can now enable a reduction in physical dimensions of the dosimeters without loss in ability to detect light output.

For the validation of optical diagnostic technologies, experimental results need to be benchmarked against the gold standard. Currently, the gold standard for tissue characterization is assessment of hematoxylin and eosin (H&E)-stained sections by a pathologist. When processing tissue into H&E sections, the shape of the tissue deforms with respect to the initial shape when it was optically measured. We demonstrate the importance of accounting for these tissue deformations when correlating optical measurement with routinely acquired histopathology. We propose a method to register the tissue in the H&E sections to the optical measurements, which corrects for these tissue deformations. We compare the registered H&E sections to H&E sections that were registered with an algorithm that does not account for tissue deformations by evaluating both the shape and the composition of the tissue and using microcomputer tomography data as an independent measure. The proposed method, which did account for tissue deformations, was more accurate than the method that did not account for tissue deformations. These results emphasize the need for a registration method that accounts for tissue deformations, such as the method presented in this study, which can aid in validating optical techniques for clinical use.

Near-infrared spectroscopy (NIRS) muscle oxygenation data are relied on in sports medicine. Many women with urinary incontinence (UI) have dysfunctional pelvic floor muscles (PFMs) but their evaluation lacks such measures; a transvaginal NIRS interface would enable the PFM to be interrogated. Paired miniature fiber-optic cables were configured on a rigid foam insert so their emitter detector arrays with an interoptode distance of 20 mm apposed the right and left inner sides of a disposable clear plastic vaginal speculum, and linked to a standard commercial NIRS instrument. Measurement capability was assessed through conduct of three maximum voluntary contractions (MVCs) and one sustained maximum voluntary contraction of the PFM with calculation of HbDiff (½RT), a validated muscle reoxygenation kinetic parameter. In all four asymptomatic controls, mean age 40, mean BMI 21.4, MVCs were associated with changes in PFM oxyhemoglobin (O₂Hb), deoxyhemoglobin (HHb) concentration, and their difference (HbDiff) comparable to those in voluntary muscle sports medicine studies. NIRS data during recovery (reoxygenation) allowed calculation of HbDiff (½RT). New techniques are called for to evaluate UI. This NIRS interface warrants further development as the provision of quantitative reoxygenation kinetics offers more comprehensive evaluation of patients with PFM dysfunction.

A multi-spectral laser speckle contrast imaging (MS-LSCI) system is proposed using only a single wavelength-swept laser, which provides both highly coherent and multi-spectral outputs to simultaneously generate laser speckle contrast images and multi-spectral images, respectively. Using a laser light swept from 770 to 821 nm at a repetition rate of 5 Hz and a CCD camera of 335 fps, 67 multi-spectral frame images are acquired in 0.76 nm wavebands over 51 nm spectral range. The spectral sub-windowing method of single wavelength-swept laser source is used to solve the lack of spectral information from a few individual light sources, which is a limitation of conventional MS-LSCI systems. In addition to the speckle flow index from the LSCI frames, the multi-spectrally encoded images can generate additional images of spectral absorbance. To further examine the performance of the MS-LSCI system, an in vivo cuff-induced ischemia experiment was conducted to show the real-time imaging of hemodynamic and blood oxygen saturation changes simultaneously over the entire 2.5  cm  ×  4.5  cm field of view.

Cerenkov radiation (CR) is the emission of UV-vis light generated by the de-excitation of the molecules in the medium, after being polarized by an excited particle traveling faster than the speed of light. When β particles travel through tissue with energies greater than 219 keV, CR occurs. Tissues possess a spectral optical window of 600 to 1100 nm. The CR within this range can be useful for quantitative preclinical studies using optical imaging and for the in-vivo evaluation of Lu177-radiopharmaceuticals (β-particle emitters). The objective of our research was to determine the experimental emission light spectrum of Lu177-CR and evaluate its transmission properties in tissue as well as the feasibility to applying CR imaging in the preclinical studies of Lu177-radiopharmaceuticals. The theoretical and experimental characterizations of the emission and transmission spectra of Lu177-CR in tissue, in the vis-NIR region (350 to 900 nm), were performed using Monte Carlo simulation and UV-vis spectroscopy. Mice Lu177-CR images were acquired using a charge-coupled detector camera and were quantitatively analyzed. The results demonstrated good agreement between the theoretical and the experimental Lu177-CR emission spectra. Preclinical CR imaging demonstrated that the biokinetics of Lu177-radiopharmaceuticals in the main organs of mice can be acquired.

Dynamic elastography is an attractive method to evaluate tissue biomechanical properties. Recently, it was extended from US- and MR-based modalities to optical ones, such as optical coherence tomography for three-dimensional (3-D) imaging of propagating mechanical waves in subsurface regions of soft tissues, such as the eye. The measured group velocity is often used to convert wave speed maps into 3-D images of the elastic modulus distribution based on the assumption of bulk shear waves. However, the specific geometry of OCE measurements in bounded materials such as the cornea and skin calls into question elasticity reconstruction assuming a simple relationship between group velocity and shear modulus. We show that in layered media the bulk shear wave assumption results in highly underestimated shear modulus reconstructions and significant structural artifacts in modulus images. We urge the OCE community to be careful in using the group velocity to evaluate tissue elasticity and to focus on developing robust reconstruction methods to accurately reconstruct images of the shear elastic modulus in bounded media.

Mueller microscopy studies of fixed unstained histological cuts of human skin models were combined with an analysis of experimental data within the framework of differential Mueller matrix (MM) formalism. A custom-built Mueller polarimetric microscope was used in transmission configuration for the optical measurements of skin tissue model adjacent cuts of various nominal thicknesses (5 to 30  μm). The maps of both depolarization and polarization parameters were calculated from the corresponding microscopic MM images by applying a logarithmic Mueller matrix decomposition (LMMD) pixelwise. The parameters derived from LMMD of measured tissue cuts and the intensity of transmitted light were used for an automated segmentation of microscopy images to delineate dermal and epidermal layers. The quadratic dependence of depolarization parameters and linear dependence of polarization parameters on thickness, as predicted by the theory, was confirmed in our measurements. These findings pave the way toward digital histology with polarized light by presenting the combination of optimal optical markers, which allows mitigating the impact of tissue cut thickness fluctuations and increases the contrast of polarimetric images for tissue diagnostics.