Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

Multi‐modal optical imaging characterization of atherosclerotic plaques

We combined cross‐polarization optical coherence tomography (CP OCT) and non‐linear microscopy based on second harmonic generation (SHG) and two‐photon‐excited fluorescence (2PEF) to assess collagen and elastin fibers and other vascular structures in the development of atherosclerosis, including identification of vulnerable plaques, which remains an important clinical problem and imaging application. CP OCT's ability to visualize tissue birefringence and cross‐scattering adds new information about the microstructure and composition of the plaque. However its interpretation can be ambiguous, because backscattering contrast may have a similar appearance to the birefringence related fringes. Our results represent a step towards minimally invasive characterization and monitoring of different stages of atherosclerosis, including vulnerable plaques. CP OCT image of intimal thickening in the human coronary artery. The dark stripe in the cross‐polarization channel (arrow) is a polarization fringe related to the phase retardation between two eigen polarization states. It is histologically located in the area of the lipid pool, however this stripe is a polarization artifact, rather than direct visualization of the lipid pool.

     

Dual‐polarization Raman spectral imaging to extract overlapping molecular fingerprints of living cells

Raman spectral imaging is gaining more and more attention in biological studies because of its label‐free characteristic. However, the discrimination of overlapping chemical contrasts has been a major challenge. In this study, we introduce an optical method to simultaneously obtain two orthogonally polarized Raman images from a single scan of the sample. We demonstrate how this technique can improve the quality and quantity of the hyperspectral Raman dataset and how the technique is expected to further extend the horizons of Raman spectral imaging in biological studies by providing more detailed chemical information.

The dual‐polarization Raman images of a HeLa cell.     

Spectrally encoded coherence tomography and reflectometry: Simultaneous en face and cross‐sectional imaging at 2 gigapixels per second

Non‐invasive biological imaging is crucial for understanding in vivo structure and function. Optical coherence tomography (OCT) and reflectance confocal microscopy are two of the most widely used optical modalities for exogenous contrast‐free, high‐resolution, three‐dimensional imaging in non‐fluorescent scattering tissues. However, sample motion remains a critical barrier to raster‐scanned acquisition and reconstruction of wide‐field anatomically accurate volumetric datasets. We introduce spectrally encoded coherence tomography and reflectometry (SECTR), a high‐speed, multimodality system for simultaneous OCT and spectrally encoded reflectance (SER) imaging. SECTR utilizes a robust system design consisting of shared optical relays, scanning mirrors, swept laser and digitizer to achieve the fastest reported in vivo multimodal imaging rate of 2 gigapixels per second. Our optical design and acquisition scheme enable spatiotemporally co‐registered acquisition of OCT cross‐sections simultaneously with en face SER images for multivolumetric mosaicking. Complementary axial and lateral translation and rotation are extracted from OCT and SER data, respectively, for full volumetric estimation of sample motion with micron spatial and millisecond temporal resolution.      

Quantitative depolarization measurements for fiber‐based polarization‐sensitive optical frequency domain imaging of the retinal pigment epithelium

A full quantitative evaluation of the depolarization of light may serve to assess concentrations of depolarizing particles in the retinal pigment epithelium and to investigate their role in retinal diseases in the human eye. Optical coherence tomography and optical frequency domain imaging use spatial incoherent averaging to compute depolarization. Depolarization depends on accurate measurements of the polarization states at the receiver but also on the polarization state incident upon and within the tissue. Neglecting this dependence can result in artifacts and renders depolarization measurements vulnerable to birefringence in the system and in the sample. In this work, we discuss the challenges associated with using a single input polarization state and traditional depolarization metrics such as the degree‐of‐polarization and depolarization power. We demonstrate quantitative depolarization measurements based on Jones vector synthesis and polar decomposition using fiber‐based polarization‐sensitive optical frequency domain imaging of the retinal pigment epithelium in a human eye.      

K‐distribution three‐dimensional mapping of biological tissues in optical coherence tomography

Probability density function (PDF) analysis with K‐distribution model of optical coherence tomography (OCT) intensity signals has previously yielded a good representation of the average number of scatterers in a coherence volume for microspheres‐in‐water systems, and has shown initial promise for biological tissue characterization. In this work, we extend these previous findings, based on single point M‐mode or two‐dimenstional slice analysis, to full three‐dimensional (3D) imaging maps of the shape parameter α of the K‐distribution PDF. After selecting a suitably sized 3D evaluation window, and verifying methodology in phantoms, the resultant parametric α images obtained in different animal tissues (rat liver and brain) show new contrasting ability not seen in conventional OCT intensity images.      

High‐definition optical coherence tomography of melanocytic skin lesions

High‐definition optical coherence tomography (HD‐OCT) scanners have recently been developed. We assessed micromorphological HD‐OCT correlates of benign naevi (BN) and malignant melanoma (MM). 28 BN and 20 MM were studied using HD‐OCT and histology. Epidermal honeycomb/cobblestone pattern, regular junctional cell nests, and edged papillae are more often observed in BN, whereas fusion of rete ridges, pagetoid cells and junctional and/or dermal nests with atypical cells are more frequently seen in MM. A high overlap of HD‐OCT features in BN and MM was observed and in 20% of MM we did not find evidence for malignancy in OCT images at all. Using HD‐OCT it is possible to visualize architectural and cellular alterations of melanocytic skin lesions. The overlap of HD‐OCT features seen in BN and MM and the absence of suspicious HD‐OCT features in some MM represents an important limitation of HD‐OCT affecting the sensitivity of HD‐OCT in diagnosing MM.

High‐definition optical coherence tomography and the corresponding vertically sectioned histology of a compound naevus.     

Dynamic single gold nanoparticle visualization by clinical intracoronary optical coherence tomography

The potential use of Gold Nanoparticles (GNPs) as contrast agents for clinical intracoronary frequency domain Optical Coherence Tomography (OCT) is here explored. The OCT contrast enhancement caused by GNPs of different sizes and morphologies has been systematically investigated and correlated with their optical properties. Among the different GNPs commercially available with plasmon resonances close to the operating wavelength of intracoronary OCT (1.3 µm), Gold Nanoshells (GNSs) have provided the best OCT contrast due to their largest scattering cross section at this wavelength. Clinical intracoronary OCT catheters are here demonstrated to be capable of three dimensional visualization and real‐time tracking of individual GNSs. Results here included open an avenue to novel application of intravascular clinical OCT in combination with GNPs, such as real time evaluation of intravascular obstructions or pressure gradients.

     

Investigating the healing mechanisms of an angiogenesis‐promoting topical treatment for diabetic wounds using multimodal microscopy

Impaired skin wound healing is a significant comorbid condition of diabetes that is caused by poor microcirculation, among other factors. Studies have shown that angiogenesis, a critical step in the wound healing process in diabetic wounds, can be promoted under hypoxia. In this study, an angiogenesis‐promoting topical treatment for diabetic wounds, which promotes angiogenesis by mimicking a hypoxic environment via inhibition of prolyl hydroxylase resulting in elevation or maintenance of hypoxia‐inducible factor, was investigated utilizing a custom‐built multimodal microscopy system equipped with phase‐variance optical coherence tomography (PV‐OCT) and fluorescence lifetime imaging microscopy (FLIM). PV‐OCT was used to track the regeneration of the microvasculature network, and FLIM was used to assess the in vivo metabolic response of mouse epidermal keratinocytes to the treatment during healing. Results show a significant decrease in the fluorescence lifetime of intracellular reduced nicotinamide adenine dinucleotide, suggesting a hypoxic‐like environment in the wounded skin, followed by a quantitative increase in blood vessel density assessed by PV‐OCT. Insights gained in these studies could lead to new endpoints for evaluation of the efficacy and healing mechanisms of wound‐healing drugs in a setting where delayed healing does not permit available methods for evaluation to take place.      

Characterization of the human myocardium by optical coherence tomography

Imaging of cardiac tissue structure plays a critical role in the treatment and understanding of cardiovascular disease. Optical coherence tomography (OCT) offers the potential to provide valuable, high‐resolution imaging of cardiac tissue. However, there is a lack of comprehensive OCT imaging data of the human heart, which could improve identification of structural substrates underlying cardiac abnormalities. The objective of this study was to provide qualitative and quantitative analysis of OCT image features throughout the human heart. Fifty human hearts were acquired, and tissues from all chambers were imaged with OCT. Histology was obtained to verify tissue composition. Statistical differences between OCT image features corresponding to different tissue types and chambers were estimated using analysis of variance. OCT imaging provided features that were able to distinguish structures such as thickened collagen, as well as adipose tissue and fibrotic myocardium. Statistically significant differences were found between atria and ventricles in attenuation coefficient, and between adipose and all other tissue types. This study provides an overview of OCT image features throughout the human heart, which can be used for guiding future applications such as OCT‐integrated catheter‐based treatments or ex vivo investigation of structural substrates.     

Optical coherence tomography follow‐up of patients treated from periodontal disease

Optical coherence tomography (OCT) is one of the most important imaging modalities for biophotonics applications. In this work, an important step towards the clinical use of OCT in dental practice is reported, by following‐up patients treated from periodontal disease (PD). A total of 147 vestibular dental sites from 14 patients diagnosed with PD were evaluated prior and after treatment, using a swept‐source OCT and two periodontal probes (Florida probe and North Carolina) for comparison. The evaluation was performed at four stages: day 0, day 30, day 60 and day 90. Exceptionally one patient was evaluated 1‐year after treatment. It was possible to visualize in the two‐dimensional images the architectural components that compose the periodontal anatomy, and identify the improvements in biofilm and dental calculus upon treatment. In the follow‐up after the treatment, it was observed in some cases decrease of the gingival thickness associated with extinction of gingival calculus. In some cases, the improvement of both depth of probing with the traditional probes and the evidence in the images of the region was emphasized. The study evidenced the ability of OCT in the identification of periodontal structures and alterations, being an important noninvasive complement or even alternative for periodontal probes for treatment follow‐up. OCT system being used in a clinical environment. Above OCT image (left) prior treatment and (right) 30 days after treatment.      

Depth‐resolved multimodal imaging: Wavelength modulated spatially offset Raman spectroscopy with optical coherence tomography

A major challenge in biophotonics is multimodal imaging to obtain both morphological and molecular information at depth. We demonstrate a hybrid approach integrating optical coherence tomography (OCT) with wavelength modulated spatially offset Raman spectroscopy (WM‐SORS). With depth colocalization obtained from the OCT, we can penetrate 1.2‐mm deep into strong scattering media (lard) to acquire up to a 14‐fold enhancement of a Raman signal from a hidden target (polystyrene) with a spatial offset. Our approach is capable of detecting both Raman and OCT signals for pharmaceutical particles embedded in turbid media and revealing the white matter at depth within a 0.6‐mm thick brain tissue layer. This depth resolved label‐free multimodal approach is a powerful route to analyze complex biomedical samples.      

One‐to‐one registration of en‐face optical coherence tomography attenuation coefficients with histology of a prostatectomy specimen

Optical coherence tomography (OCT), enables high‐resolution 3D imaging of the morphology of light scattering tissues. From the OCT signal, parameters can be extracted and related to tissue structures. One of the quantitative parameters is the attenuation coefficient; the rate at which the intensity of detected light decays in depth. To couple the quantitative parameters with the histology one‐to‐one registration is needed. The primary aim of this study is to validate a registration method of quantitative OCT parameters to histological tissue outcome through one‐to‐one registration of OCT with histology. We matched OCT images of unstained fixated prostate tissue slices with corresponding histology slides, wherein different histologic types were demarcated. Attenuation coefficients were determined by a supervised automated exponential fit (corrected for point spread function and sensitivity roll‐off related signal losses) over a depth of 0.32 mm starting from 0.10 mm below the automatically detected tissue edge. Finally, the attenuation coefficients corresponding to the different tissue types of the prostate were compared. From the attenuation coefficients, we produced the squared relative residue and goodness‐of‐fit metric R². This article explains the method to perform supervised automated quantitative analysis of OCT data, and the one‐to‐one registration of OCT extracted quantitative data with histopathological outcomes.      

Origin of improved depth penetration in dual‐axis optical coherence tomography: a Monte Carlo study

Recent studies have demonstrated that extended imaging depth can be achieved using dual‐axis optical coherence tomography (DA‐OCT). By illuminating and collecting at an oblique angle, multiple forward scattered photons from large probing depths are preferentially detected. However, the mechanism behind the enhancement of imaging depth needs further illumination. Here, the signal of a DA‐OCT system is studied using a Monte Carlo simulation. We modeled light transport in tissue and recorded the spatial and angular distribution of photons exiting the tissue surface. Results indicate that the spatial separation and offset angle created by the non‐telecentric scanning configuration promote the collection of more deeply propagating photons than conventional on‐axis OCT.      

Identification and characterization of bladder cancer by low‐resolution fiber‐optic Raman spectroscopy

Raman spectroscopy has been proved to be a promising diagnostic technique for various cancers detection. A major drawback for its clinical translation is the intrinsic weakness of Raman effects. Highly sensitive equipment and optimal measurement conditions are generally applied to overcome this drawback. However, these equipment are usually bulky, expensive and may also be easily influenced by surrounding environment. In this preliminary work, a low‐resolution fiber‐optic Raman sensing system is applied to evaluate the diagnostic potential of Raman spectroscopy to identify different bladder pathologies ex vivo. A total number of 262 spectra taken from 32 bladder specimens are included in this study. These spectra are categorized into 3 groups by histopathological analysis, namely normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors. Principal component analysis fed artificial neural network are used to train a classification model for the spectral data with 10‐fold cross‐validation and an overall prediction accuracy of 93.1% is obtained. The sensitivities and specificities for normal bladder tissues, low‐grade bladder tumors and high‐grade bladder tumors are 88.5% and 95.1%, 90.3% and 98%, and 97.5% and 96.4%, respectively. These results demonstrate the potential of using a low‐resolution fiber‐optic Raman system for in vivo bladder cancer diagnosis.      

Polarization‐multiplexed,dual‐beam swept source optical coherence tomography angiography

A polarization‐multiplexed, dual‐beam setup is proposed to expand the field of view (FOV) for a swept source optical coherence tomography angiography (OCTA) system. This method used a Wollaston prism to split sample path light into 2 orthogonal‐polarized beams. This allowed 2 beams to shine on the cornea at an angle separation of ~14°, which led to a separation of ~4.2 mm on the retina. A 3‐mm glass plate was inserted into one of the beam paths to set a constant path length difference between the 2 polarized beams so the interferogram from the 2 beams are coded at different frequency bands. The resulting OCTA images from the 2 beams were coded with a depth separation of ~2 mm. A total of 5 × 5 mm² angiograms from the 2 beams were obtained simultaneously in 4 seconds. The 2 angiograms then were montaged to get a wider FOV of ~5 × 9.2 mm².      

Hyperspectral optical coherence tomography for in vivo visualization of melanin in the retinal pigment epithelium

Previous studies for melanin visualization in the retinal pigment epithelium (RPE) have exploited either its absorption properties (using photoacoustic tomography or photothermal optical coherence tomography [OCT]) or its depolarization properties (using polarization sensitive OCT). However, these methods are only suitable when the melanin concentration is sufficiently high. In this work, we present the concept of hyperspectral OCT for melanin visualization in the RPE when the concentration is low. Based on white light OCT, a hyperspectral stack of 27 wavelengths (440‐700 nm) was created in post‐processing for each depth‐resolved image. Owing to the size and shape of the melanin granules in the RPE, the variations in backscattering coefficient as a function of wavelength could be identified—a result which is to be expected from Mie theory. This effect was successfully identified both in eumelanin‐containing phantoms and in vivo in the low‐concentration Brown Norway rat RPE.      

TiO2‐coated fluoride nanoparticles for dental multimodal optical imaging

Core‐shell nanostructures associated with photonics techniques have found innumerous applications in diagnostics and therapy. In this work, we introduce a novel core‐shell nanostructure design that serves as a multimodal optical imaging contrast agent for dental adhesion evaluation. This nanostructure consists of a rare‐earth‐doped (NaYF₄:Yb 60%, Tm 0.5%)/NaYF₄ particle as the core (hexagonal prism, ~51 nm base side length) and the highly refractive TiO₂ material as the shell (~thickness of 15 nm). We show that the TiO₂ shell provides enhanced contrast for optical coherence tomography (OCT), while the rare‐earth‐doped core upconverts excitation light from 975 nm to an emission peaked at 800 nm for photoluminescence imaging. The OCT and the photoluminescence wide‐field images of human tooth were demonstrated with this nanoparticle core‐shell contrast agent. In addition, the described core‐shell nanoparticles (CSNps) were dispersed in the primer of a commercially available dental bonding system, allowing clear identification of dental adhesive layers with OCT. We evaluated that the presence of the CSNp in the adhesive induced an enhancement of 67% scattering coefficient to significantly increase the OCT contrast. Moreover, our results highlight that the upconversion photoluminescence in the near‐infrared spectrum region is suitable for image of deep dental tissue.      

Intravascular magnetomotive optical coherence tomography of targeted early‐stage atherosclerotic changes in ex vivo hyperlipidemic rabbit aortas

We report the development of an intravascular magnetomotive optical coherence tomography (IV‐MM‐OCT) system used with targeted protein microspheres to detect early‐stage atherosclerotic fatty streaks/plaques. Magnetic microspheres (MSs) were injected in vivo in rabbits, and after 30 minutes of in vivo circulation, excised ex vivo rabbit aorta samples specimens were then imaged ex vivo with our prototype IV‐MM‐OCT system. The alternating magnetic field gradient was provided by a unique pair of external custom‐built electromagnetic coils that modulated the targeted magnetic MSs. The results showed a statistically significant MM‐OCT signal from the aorta samples specimens injected with targeted MSs.

Representative magnetomotive signal (green) using targeted and non‐targeted magnetomotive microspheres in atherosclerotic diseased rabbit aortas.     

Hand‐held,clinical dual mode ultrasound ‐ photoacoustic imaging of rat urinary bladder and its applications

Urinary bladder imaging is critical to diagnose urinary tract disorders, and bladder cancer. There is a great need for safe, non‐invasive, and sensitive imaging technique which enables bladder imaging. Photoacoustic imaging is a rapidly growing imaging technique for various biological applications. It can be combined with clinical ultrasound imaging system for hand‐held, dual modal ultrasound‐photoacoustic real‐time imaging. Structural (bladder wall) and functional (accretion of nanoparticles) bladder imaging is shown here with combined ultrasound and photoacoustic imaging in rats. Photoacoustic images of bladder wall is shown using black ink as the contrast agent. Chicken tissues were stacked on the abdomen of the animal to demonstrate the feasibility of photoacoustic imaging till a depth of 2 cm. Also, the feasibility of photoacoustic imaging for a common bladder disorder, vesicoureteral reflux is studied using urinary tract mimicking phantom. It is also shown that a clinical ultrasound system can be used for photoacoustic imaging of non‐invasive clearance study of gold nanorods from circulation by monitoring the gradual accumulation of the gold nanorods in the bladder. The time taken for accumulation of nanorods in the bladder can be used as an indicator of the clearance rate of the nanoparticle circulation from the body.