Optical coherence tomography for ultrahigh resolution in vivo imaging

Optical coherence tomography (OCT) is an emerging biomedical optical imaging technique that performs high-resolution, cross-sectional tomographic imaging of microstructure in biological systems. OCT can achieve image resolutions of 1-15 microm, one to two orders of magnitude finer than standard ultrasound. The image penetration depth of OCT is determined by the optical scattering and is up to 2-3 mm in tissue. OCT functions as a type of 'optical biopsy' to provide cross-sectional images of tissue structure on the micron scale. It is a promising imaging technology because it can provide images of tissue in situ and in real time, without the need for excision and processing of specimens.     

In vivo biomicroscopy of the skin with high-resolution magnetic resonance imaging and high frequency ultrasound.

Noninvasive imaging and characterization of the skin is of great interest in dermatology. In order to get relevant diagnostic information, high-resolution imaging techniques have to be applied. Ultrasonic imaging is a potential method for this purpose where the special requirements concerning the spatial resolution make it essential to apply high frequency ultrasound (HFUS). Alternatively, magnetic resonance imaging (MRI), being a very promising imaging modality, also shows the perspective of becoming a valuable diagnostic tool in dermatology. However, to account for the small dimensions of the structures under observation, very specialized system designs have to be developed. In this paper, a HFUS imaging system working in the 50 MHz and 100 MHz range is applied for high-resolution skin imaging. Furthermore, a commercial MRI-system was equipped with specially designed low noise rf (radio frequency) coils with minimized volume, and customized imaging sequences were applied to optimize the signal-to-noise ratio. With HFUS and high-resolution magnetic resonance (HR-MR) imaging complementary imaging techniques for in vivo biomicroscopy of the skin are available.     

Static and dynamic imaging of alveoli using optical coherence tomography needle probes

Imaging of alveoli in situ has for the most part been infeasible due to the high resolution required to discern individual alveoli and limited access to alveoli beneath the lung surface. In this study, we present a novel technique to image alveoli using optical coherence tomography (OCT). We propose the use of OCT needle probes, where the distal imaging probe has been miniaturized and encased within a hypodermic needle (as small as 30-gauge, outer diameter 310 μm), allowing insertion deep within the lung tissue with minimal tissue distortion. Such probes enable imaging at a resolution of ～12 μm within a three-dimensional cylindrical field of view with diameter ～1.5 mm centered on the needle tip. The imaging technique is demonstrated on excised lungs from three different species: adult rats, fetal sheep, and adult pigs. OCT needle probes were used to image alveoli, small bronchioles, and blood vessels, and results were matched to histological sections. We also present the first dynamic OCT images acquired with an OCT needle probe, allowing tracking of individual alveoli during simulated cyclical lung inflation and deflation.     

Ex vivo optical coherence tomography and laser-induced fluorescence spectroscopy imaging of murine gastrointestinal tract

Optical coherence tomography (OCT) and laser-induced fluorescence (LIF) spectroscopy each have clinical potential in identifying human gastrointestinal (GI) pathologies, yet their diagnostic capability in mouse models is unknown. In this study, we combined the 2 modalities to survey the GI tract of a variety of mouse strains and ages and to sample dysplasias and inflammatory bowel disease (IBD) of the intestines. Segments (length, 2.5 cm) of duodenum and lower colon and the entire esophagus were imaged ex-vivo with combined OCT and LIE We evaluated 30 normal mice (A/J and 10- and 21-wk-old and retired breeder C57BL/6J) and 10 mice each of 2 strains modeling colon cancer and IBD (Apc(Min) and IL2-deficient mice, respectively). Histology was used to classify tissue regions as normal, Peyer patch, dysplasia, adenoma, or IBD. Features in corresponding OCT images were analyzed. Spectra from each category were averaged and compared via Student t tests. OCT provided structural information that led to identification of the imaging characteristics of healthy mouse GI. With histology as the 'gold standard,' we developed preliminary image criteria for early disease in the form of adenomas, dysplasias, and IBD. LIF characterized the endogenous fluorescence of mouse GI tract, with spectral features corresponding to collagen, NADH, and hemoglobin. In the IBD sample, LIF emission spectra displayed potentially diagnostic peaks at 635 and 670 nm, which we attributed to increased porphyrin production by bacteria associated with IBD. OCT and LIF appear to be useful and complementary modalities for ex vivo imaging of mouse GI tissues.     

Real-time imaging of rabbit retina with retinal degeneration by using spectral-domain optical coherence tomography

Background

Recently, a transgenic rabbit with rhodopsin Pro 347 Leu mutation was generated as a model of retinitis pigmentosa (RP), which is characterized by a gradual loss of vision due to photoreceptor degeneration. The purpose of the current study is to noninvasively visualize and assess time-dependent changes in the retinal structures of a rabbit model of retinal degeneration by using speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT).

Methodology/Principal Findings

Wild type (WT) and RP rabbits (aged 4–20 weeks) were investigated using SD-OCT. The total retinal thickness in RP rabbits decreased with age. The thickness of the outer nuclear layer (ONL) and between the external limiting membrane and Bruch''s membrane (ELM–BM) were reduced in RP rabbits around the visual streak, compared to WT rabbits even at 4 weeks of age, and the differences increased with age. However, inner nuclear layer (INL) thickness in RP rabbits did not differ from that of WT during the observation period. The ganglion cell complex (GCC) thickness in RP rabbits increased near the optic nerve head but not around the visual streak in the later stages of the observation period. Hyper-reflective change was widely observed in the inner segments (IS) and outer segments (OS) of the photoreceptors in the OCT images of RP rabbits. Ultrastructural findings in RP retinas included the appearance of small rhodopsin-containing vesicles scattered in the extracellular space around the photoreceptors.

Conclusions/Significance

In the current study, SD-OCT provided the pattern of photoreceptor degeneration in RP rabbits and the longitudinal changes in each retinal layer through the evaluation of identical areas over time. The time-dependent changes in the retinal structure of RP rabbits showed regional and time-stage variations. In vivo imaging of RP rabbit retinas by using SD-OCT is a powerful method for characterizing disease dynamics and for assessing the therapeutic effects of experimental interventions.     

Heterogeneous pattern of retinal nerve fiber layer in multiple sclerosis. High resolution optical coherence tomography: potential and limitations

Background

Recently the reduction of the retinal nerve fibre layer (RNFL) was suggested to be associated with diffuse axonal damage in the whole CNS of multiple sclerosis (MS) patients. However, several points are still under discussion. (1) Is high resolution optical coherence tomography (OCT) required to detect the partly very subtle RNFL changes seen in MS patients? (2) Can a reduction of RNFL be detected in all MS patients, even in early disease courses and in all MS subtypes? (3) Does an optic neuritis (ON) or focal lesions along the visual pathways, which are both very common in MS, limit the predication of diffuse axonal degeneration in the whole CNS? The purpose of our study was to determine the baseline characteristics of clinical definite relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients with high resolution OCT technique.

Methodology

Forty-two RRMS and 17 SPMS patients with and without history of uni- or bilateral ON, and 59 age- and sex-matched healthy controls were analysed prospectively with the high resolution spectral-domain OCT device (SD-OCT) using the Spectralis 3.5mm circle scan protocol with locked reference images and eye tracking mode. Furthermore we performed tests for visual and contrast acuity and sensitivity (ETDRS, Sloan and Pelli-Robson-charts), for color vision (Lanthony D-15), the Humphrey visual field and visual evoked potential testing (VEP).

Principal Findings

All 4 groups (RRMS and SPMS with or without ON) showed significantly reduced RNFL globally, or at least in one of the peripapillary sectors compared to age-/sex-matched healthy controls. In patients with previous ON additional RNFL reduction was found. However, in many RRMS patients the RNFL was found within normal range. We found no correlation between RNFL reduction and disease duration (range 9–540 months).

Conclusions

RNFL baseline characteristics of RRMS and SPMS are heterogeneous (range from normal to markedly reduced levels).     

Magnetic and contrast properties of labeled platelets for magnetomotive optical coherence tomography

This article introduces a new functional imaging paradigm that uses optical coherence tomography (OCT) to detect rehydrated, lyophilized platelets (RL platelets) that are in the preclinical trial stage and contain superparamagnetic iron oxides (SPIOs) approved by the U.S. Food and Drug Administration. Platelets are highly functional blood cells that detect and adhere to sites of vascular endothelial damage by forming primary hemostatic plugs. By applying magnetic gradient forces, induced nanoscale displacements (magnetomotion) of the SPIO-RL platelets are detected as optical phase shifts in OCT. In this article, we characterize the iron content and magnetic properties of SPIO-RL platelets, construct a model to predict their magnetomotion in a tissue medium, and demonstrate OCT imaging in tissue phantoms and ex vivo pig arteries. Tissue phantoms containing SPIO-RL platelets exhibited >3 dB contrast/noise ratio at ≥1.5 × 10⁹ platelets/cm³. OCT imaging was performed on ex vivo porcine arteries after infusion of SPIO-RL platelets, and specific contrast was obtained on an artery that was surface-damaged (P < 10⁻⁶). This may enable new technologies for in vivo monitoring of the adherence of SPIO-RL platelets to sites of bleeding and vascular damage, which is broadly applicable for assessing trauma and cardiovascular diseases.     

Measurements of donor endothelial keratoplasty lenticules prepared from fresh donated whole eyes by using ultrasound and optical coherence tomography

This study was conducted to analyze the profile and thickness of endothelial keratoplasty lenticules prepared from fresh donated whole eyes with Visante optical coherence tomography (V-OCT) compared to measurements obtained from ultrasound pachymetry (USP) at the Central Eye Bank of Iran. Microkeratome-assisted precut corneas were prepared for Descemet stripping automated endothelial keratoplasty by using standard eye bank protocol. Central posterior lenticule thickness (CPLT) on fresh whole eye, before excising corneoscleral disc and transferring to Optisol-GS, was measured by USP. V-OCT was used to measure central, paracentral, and midperipheral thicknesses of lenticules after transferring the tissue to Optisol-GS. Chi Square and Bonferroni tests were respectively used to uncover the differences between the USP and V-OCT measurements and also the thickness profile of lenticules. Postoperative reports for the entire transplanted lenticules were recorded. Accordingly, on evaluation of 312 enrolled precut corneas, CPLT measurements by V-OCT versus USP were statistically different (mean: 136 µm vs 165 µm, respectively; P = 0.008). Thickness profile of the posterior lenticules revealed increased thickness from the central to the peripheral parts of the cornea (mean increase of 16 µm at the pericentral and 64.2 µm at the peripheral locations, respectively); however, the increase in the thickness was relatively symmetrical. Postoperative reports of transplanted lenticules were unremarkable, since there were no posterior flap detachments. In essence, V-OCT measurements of microkeratome-assisted precut lenticules prepared from fresh donated whole eyes averaged 29 μm thinner than USP measurements and revealed a significant but symmetric increase of thickness towards the peripheral parts of the corneas. However, the variation in the thickness profile did not affect the attachment or the clarity of transplanted precut lenticlues.     

Four‐dimensional imaging of murine subpleural alveoli using high‐speed optical coherence tomography

The investigation of lung dynamics on alveolar scale is crucial for the understanding and treatment of lung diseases, such as acute lung injury and ventilator induced lung injury, and to promote the development of protective ventilation strategies. One approach to this is the establishment of numerical simulations of lung tissue mechanics where detailed knowledge about three‐dimensional alveolar structure changes during the ventilation cycle is required. We suggest four‐dimensional optical coherence tomography (OCT) imaging as a promising modality for visualizing the structural dynamics of single alveoli in subpleural lung tissue with high temporal resolution using a mouse model. A high‐speed OCT setup based on Fourier domain mode locked laser technology facilitated the acquisition of alveolar structures without noticeable motion artifacts at a rate of 17 three‐dimensional stacks per ventilation cycle. The four‐dimensional information, acquired in one single ventilation cycle, allowed calculating the volume‐pressure curve and the alveolar compliance for single alveoli. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

A review of high-resolution X-ray computed tomography and other imaging modalities for small animal research

Dedicated high-resolution small animal systems have recently emerged as important new tools for laboratory animal research. These imaging systems permit researchers to noninvasively screen animal models for mutations or pathologies and to monitor disease progression and response to therapy. The authors survey various small animal imaging modalities, including MRI, PET, SPECT, and microCT, and discuss several representative microCT mouse imaging studies.     

Dependence of optical attenuation coefficient and mechanical tension of irradiated human cartilage measured by optical coherence tomography

As banked human tissues are not widely available, the development of new non-destructive and contactless techniques to evaluate the quality of allografts before distribution for transplantation is very important. Also, tissues will be processed accordingly to standard procedures and to minimize disease transmission most tissue banks will include a decontamination or sterilization step such as ionizing radiation. In this work, we present a new method to evaluate the internal structure of frozen or glycerol processed human cartilages, submitted to various dosis of irradiation, using the total optical attenuation coefficient retrieved from optical coherence tomography (OCT) images. Our results show a close relationship between tensile properties and the total optical attenuation coefficient of cartilages. Therefore, OCT associated with the total optical attenuation coefficient open a new window to evaluate quantitatively biological changes in processed tissues.     

HiSStology: high spectral and spatial resolution magnetic resonance imaging detection of vasculature validated by histology and micro-computed tomography

High spectral and spatial resolution (HiSS) data, acquired with echo-planar spectroscopic imaging (EPSI), can be used to acquire water spectra from each small image voxel. These images are sensitive to changes in local susceptibility caused by superparamagnetic iron oxide particles (SPIO); therefore, we hypothesized that images derived from HiSS data are very sensitive to tumor neovasculature following injection of SPIO. Accurate image registration was used to validate HiSS detection of neovasculature with histology and micro-computed tomographic (microCT) angiography. Athymic nude mice and Copenhagen rats were inoculated with Dunning AT6.1 prostate tumor cells in the right hind limb. The tumor region was imaged pre- and post-intravenous injection of SPIO. Three-dimensional assemblies of the CD31-stained histologic slices of the mouse legs and the microCT images of the rat vascular casts were registered with EPSI. The average distance between HiSS-predicted regions of high vascular density on magnetic resonance imaging and CD31-stained regions on histology was 200 μm. Similarly, vessels identified by HiSS in the rat images coincided with vasculature in the registered microCT image. The data demonstrate a strong correlation between tumor vasculature identified using HiSS and two gold standards: histology and microCT angiography.     

A simple method for high temporal resolution calcium imaging with dual excitation dyes.

Calcium-sensitive dual excitation dyes, such as fura-2, are now widely used to measure the free calcium concentration ([Ca2+]) in living cells. Preferentially, [Ca2+] is calculated in a ratiometric manner, but if calcium images need to be acquired at high temporal resolution, a potential drawback of ratiometry is that it requires equally fast switching of the excitation light between two wavelengths. To circumvent continuous excitation switching, some investigators have devised methods for calculating [Ca2+] from single-wavelength measurements combined with the acquisition of a single ratiometric pair of fluorescence images at the start of the recording. These methods, however, are based on the assumption that the concentration of the dye does not change during the experiment, a condition that is often not fulfilled. We describe here a method of single-wavelength calcium imaging, in which the dye concentration is estimated from ratiometric fluorescence image pairs acquired at regular intervals during the recording period, that furthermore includes a correction for the changing dye concentration in the calculation of [Ca2+].     

The impact of utilizing different optical coherence tomography devices for clinical purposes and in multiple sclerosis trials

Optical coherence tomography (OCT) derived retinal measures, particularly peri-papillary retinal nerve fiber layer (RNFL) thickness, have been proposed as outcome measures in remyelinating and neuroprotective trials in multiple sclerosis (MS). With increasing utilization of multiple centers to improve power, elucidation of the impact of different OCT technologies is crucial to the design and interpretation of such studies. In this study, we assessed relation and agreement between RNFL thickness and total macular volume (in MS and healthy controls) derived from three commonly used OCT devices: Stratus time-domain OCT, and Cirrus HD-OCT and Spectralis, two spectral-domain (SD) OCT devices. OCT was performed on both Cirrus HD-OCT and Stratus in 229 participants and on both Cirrus HD-OCT and Spectralis in a separate cohort of 102 participants. Pearson correlation and Bland-Altman analyses were used to assess correlation and agreement between devices. All OCT retinal measures correlated highly between devices. The mean RNFL thickness was 7.4 μm lower on Cirrus HD-OCT than Stratus, indicating overall poor agreement for this measurement between these machines. Further, the limits of agreement (LOA) between Cirrus HD-OCT and Stratus were wide (-4.1 to 18.9 μm), indicating poor agreement at an individual subject level. The mean RNFL thickness was 1.94 μm (LOA: -5.74 to 9.62 μm) higher on Spectralis compared to Cirrus HD-OCT, indicating excellent agreement for this measurement across this cohort. Although these data indicate that these three devices agree poorly at an individual subject level (evidenced by wide LOA in both study cohorts) precluding their co-utilization in everyday practice, the small difference for mean measurements between Cirrus HD-OCT and Spectralis indicate pooled results from these two SD-devices could be used as outcome measures in clinical trials, provided patients are scanned on the same machine throughout the trial, similar to the utilization of multiple different MRI platforms in MS clinical trials.     

Comparison of treatment modalities for recurrent basal cell carcinoma.

We studied 97 patients with recurrent basal cell carcinomas; 20 were treated by surgery, 35 by radiation, 40 with chemosurgery, and two patients had no treatment (because their lesions were too far advanced). There was a treatment failure rate of 11 percent for radiation therapy, 12 percent for chemosurgery and 5 percent for surgical excision. Recurrent BCC requires a careful selection of the mode of therapy, aggressive treatment, and careful long-term follow-up. Using these principles, an excellent cure rate can be achieved by all the 3 modes of therapy included in this report.     

Synthesis and functional characterization of a fluorescent peptide probe for non invasive imaging of collagen in live tissues

Targeted molecular imaging to detect changes in the structural and functional organization of tissues, at the molecular level, is a promising approach for effective and early diagnosis of diseases. Quantitative and qualitative changes in type I collagen, which is a major component in the extra cellular matrix (ECM) of skin and other vital organs like lung, liver, heart and kidneys, are often associated with the pathophysiology of these organs. We have synthesized a fluorescent probe that comprises collagelin, a specific collagen binding peptide, coupled to fluorescent porphyrin that can effectively detect abnormal deposition of collagen in live tissues by emitting fluorescence in the near infra red (NIR) region. In this report we have presented the methodology for coupling of 5-(4-carboxy phenyl)-10, 15, 20-triphenyl porphyrin (C-TPP) to the N-terminal of collagelin or to another mutant peptide (used as a control). We have evaluated the efficacy of these fluorescent peptides to detect collagen deposition in live normal and abnormal tissues. Our results strongly suggest that porphyrin-tagged collagelin can be used as an effective probe for the non invasive in vivo detection of tissue fibrosis, especially in the liver.     

Macular thickness and volume parameters measured using optical coherence tomography (OCT) for evaluation of glaucoma patients

The aim of this study was to evaluate macular thickness parameters in glaucoma patients and to compare them to normal subjects using Optical Coherence Tomography (OCT). This prospective, observational study included 20 primary open angle glaucoma patients (POAG) and 20 healthy subjects in control group. Exclusion criteria were diabetes and other macular pathology, like age-related macular degeneration, macular oedema, central serous retinopathy and high myopia >4.00 dsph. OCT imaging of peripapillar retina and macular area were performed using Cirrus HD OCT In these two groups of patients we analyzed changes of macular thickness parameters (central subfield thickness, macular volume, and average macular thickness). The group of glaucoma patients had decreased values of the two macular thickness parameters: macular volume and average macular thickness, compared to control group. There was no difference in central macular thickness, presumably because of the absence of the ganglion cells in this layer. Macular imaging can be a useful additional method to determine glaucoma status and has a potential for tracking glaucoma progression.     

Laser hyperthermia of tumors using gold nanoparticles monitored by optical coherence tomography and acoustic thermometry

Local laser hyperthermia of grafted RShM-5 tumors in mice with the use of plasmon resonant gold nanoparticles has been carried out. Accumulation of particles in the tumor was monitored in vivo noninvasively by optical coherence tomography. Thereby it was determined that the maximal content of nanoparticles in the tumor was reached within 5 h after intravenous administration, and laser hyperthermia was performed at this time. Monitoring the tumor temperature during the treatment by IR thermography and acoustic thermometry showed that the nanoparticles provided efficient temperature elevation inside the tumor as well as more selective heating. Local laser hyperthermia with gold nanoparticles, but not the laser exposure alone, substantially inhibited tumor growth in several days after a single session.