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)     

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.     

Quantifying dental biofilm growth using cross-polarization optical coherence tomography

Aims: Quantifying the ex vivo growth of complex multispecies dental biofilms using cross‐polarization 1310‐nm optical coherence tomography (CP‐OCT) system was investigated. Methods and Results: Bacterial microcosms, which were derived from plaque samples of paediatric subjects, were incubated in a biofilm reactor system containing discs of different dental materials for 72 h with daily sucrose pulsing (5×). CP‐OCT analysis of biofilm mass was validated with crystal violet (CV) assays at various growth stages of these complex biofilms. CP‐OCT was able to filter out the back‐reflected signals of water layers in the hydrated biofilm and allowed for direct biofilm quantification. The overall depth‐resolved scattering intensity of the biofilm showed very strong positive correlation with CV assay quantification (Spearman’s ρ = 0·92) during the growth phase of the biofilm. Conclusion: CP‐OCT was able to quantify the mass of the biofilm by measuring the overall depth‐resolved scattering of the biofilm. Significance and Impact of the Study: CP‐OCT has the ability to nondestructively monitor biofilm growth and elucidate the growth characteristics of these microcosms on different dental material compositions.     

Imaging growth dynamics of tumour spheroids using optical coherence tomography

Optical coherence tomography (OCT) was used to monitor the dynamics of tumour spheroid formation by the hanging drop method. In contrast to microscopy, the estimates obtained using OCT for the volume of the spheroid, were consistent with the measured changes in cell number as a function of time. The OCT images also revealed heterogeneous structures in the spheroids of ∼200 μm diameter. These corresponded to the necrotic regions identified by fluorescence of propidium iodide stained cells.     

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.     

Influence of osmolytes on in vivo glucose monitoring using optical coherence tomography

Diabetes mellitus and its complications are the third leading cause of death in the world, exceeded only by cardiovascular disease and cancer. Tighter monitoring and control of blood glucose could minimize complications associated with diabetes. Recently, optical coherence tomography (OCT) for noninvasive glucose monitoring was proposed and tested in vivo. The aim of this work was to investigate the influence of changes in blood glucose concentration ([glu]) and sodium concentration ([Na+]) on the OCT signal. We also investigated the influence of other important analytes on the sensitivity of glucose monitoring with OCT. The experiments were carried out in anesthetized female pigs. The OCT images were acquired continuously from skin, while [glu] and [Na+] were experimentally varied within their physiological ranges. Correlations of the OCT signal slope with [glu] and [Na+] were studied at different tissue depths. The tissue area probed with OCT was marked and cut for histological examination. The correlation of blood [glu] and [Na+] with the OCT signal slope was observed in separate tissue layers. On average, equimolar changes in [glu] produced 2.26 +/- 1.15 greater alterations of the OCT signal slope than changes in [Na+]. Variation of concentrations of other analytes did not influence the OCT signal slope. The influence of [Na+] on relative changes in the OCT signal slope was generally less than [glu]-induced changes. OCT is a promising method for noninvasive glucose monitoring because of its ability to track the influence of changing [glu] on individual tissue layers.     

Noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes

We have generated a series of quenched near-infrared fluorescent activity-based probes (qNIRF-ABPs) that covalently target the papain-family cysteine proteases shown previously to be important in multiple stages of tumorigenesis. These 'smart' probes emit a fluorescent signal only after covalently modifying a specific protease target. After intravenous injection of NIRF-ABPs into mice bearing grafted tumors, noninvasive, whole-body imaging allowed direct monitoring of cathepsin activity. Importantly, the permanent nature of the probes also allowed secondary, ex vivo biochemical profiling to identify specific proteases and to correlate their activity with whole-body images. Finally, we demonstrate that these probes can be used to monitor small-molecule inhibition of protease targets both biochemically and by direct imaging methods. Thus, NIRF-ABPs are (i) potentially valuable new imaging agents for disease diagnosis and (ii) powerful tools for preclinical and clinical testing of small-molecule therapeutic agents in vivo.     

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.     

Imaging atherosclerotic plaque composition with intracoronary optical coherence tomography

Optical coherence tomography (OCT) allows highly accurate diagnosis of atherosclerotic plaques, including measurement of the thickness of fibrous caps, permitting an assessment of the risk of rupture. While the OCT image presents morphological information in highly resolved detail, it relies on interpretation by trained readers for the identification of tissue type. We developed a method for quantitative classification of atherosclerotic plaque constituents. The optical attenuation coefficient μ_t distinguishes different tissue types: necrotic core and macrophage infiltration exhibit strong attenuation, μ_t≥10 mm⁻¹, while calcific and fibrous tissue have a lower μ_t≈2–5 mm⁻¹. (Neth Heart J 2009;17:448-50.)     

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.     

Doppler optical coherence tomography for measuring flow in engineered tissue

The engineering of human tissue represents a major paradigm shift in clinical medicine. Early embodiments of tissue engineering are currently being taken forward to the clinic by production methods that are essentially extensions of laboratory manual procedures. However, to achieve the status of routine large-scale clinical practice, automation and scale-out processes are required. This in turn will require the development of reliable on-line monitoring and control systems. This paper examines one demand of crucial importance, namely the real time in vitro monitoring of the flow characteristics through growing tissue since this has a complex interrelationship. Doppler optical coherence tomography (DOCT) is a recently developed imaging technique for studying the rheological properties of tissues in vivo. Capable of non-invasive imaging in real time with high resolution, it is potentially ideal for the continuous monitoring of engineered tissues in vitro. As a base line, the current status of DOCT in vivo is therefore reviewed. This paper also reports the first preliminary use of DOCT in tissue engineering. The application described involves the imaging of a fully developed laminar flow through a combined tissue fabrication/bioreactor with a tissue-engineered construct (substitute blood vessel) in situ.     

Comparison of high frequency ultrasound and optical coherence tomography as modalities for high resolution and non invasive skin imaging.

High frequency ultrasound (HFUS) and optical coherence tomography (OCT) are techniques for high resolution imaging of tissues. The penetration depth of these modalities is limited, but it is sufficiently large enough for non invasive skin imaging. HFUS and OCT are based on the same concept. Waves (ultrasonic waves, respectively light waves) propagate along a narrow beam, are backscattered at tissue inhomogeneities and analyzed over time of flight to obtain spatially resolved morphological information. The objective of this paper is to compare HFUS and OCT in terms of resolution, dynamic range and contrast and to assess their value as tools for high resolution skin imaging. Measurements on phantoms and in vivo have been performed with a 100 MHz ultrasound system and an OCT-scanner working in the near infrared spectrum at 1300 nm wave-length. From the measurements, it can be concluded that OCT delivers an almost isotropic resolution (axial resolution about 5.8 microns, lateral resolution about 4.1 microns), whereas the resolution of the investigated HFUS system is more anisotropic (axial resolution about 9.3 microns, lateral resolution about 60 microns). HFUS and OCT show different penetration depths and a different contrast. Both techniques can, therefore, be combined advantageously in a multimodality approach to account for their individual characteristics.     

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.     

Noninvasive cross-sectional observation of three-dimensional cell sheet-tissue-fabrication by optical coherence tomography

Cell sheet engineering allows investigators/clinicians to prepare cell-dense three-dimensional (3-D) tissues, and various clinical trials with these fabricated tissues have already been performed for regenerating damaged tissues. Cell sheets are easily manipulated and 3-D tissues can be rapidly fabricated by layering the cell sheets. This study used optical coherence tomography (OCT) to noninvasively analyze the following processes: (1) adhesions between layered cell sheets, and (2) the beating and functional interaction of cardiac cell sheet-tissues for fabricating functional thicker 3-D tissues. The tight adhesions and functional couplings between layered cell sheets could be observed cross-sectionally and in real time. Importantly, the noninvasive and cross-sectional analyses of OCT make possible to fabricate 3-D tissues by confirming the adherence and functional couplings between layered cell sheets. OCT technology would contribute to cell sheet engineering and regenerative medicine.     

Arrhythmia caused by a Drosophila tropomyosin mutation is revealed using a novel optical coherence tomography instrument

Background

Dilated cardiomyopathy (DCM) is a severe cardiac condition that causes high mortality. Many genes have been confirmed to be involved in this disease. An ideal system with which to uncover disease mechanisms would be one that can measure the changes in a wide range of cardiac activities associated with mutations in specific, diversely functional cardiac genes. Such a system needs a genetically manipulable model organism that allows in vivo measurement of cardiac phenotypes and a detecting instrument capable of recording multiple phenotype parameters.

Methodology and Principal Findings

With a simple heart, a transparent body surface at larval stages and available genetic tools we chose Drosophila melanogaster as our model organism and developed for it a dual en-face/Doppler optical coherence tomography (OCT) instrument capable of recording multiple aspects of heart activity, including heart contraction cycle dynamics, ostia dynamics, heartbeat rate and rhythm, speed of heart wall movement and light reflectivity of cardiomyocytes in situ. We applied this OCT instrument to a model of Tropomyosin-associated DCM established in adult Drosophila. We show that DCM pre-exists in the larval stage and is accompanied by an arrhythmia previously unidentified in this model. We also detect reduced mobility and light reflectivity of cardiomyocytes in mutants.

Conclusion

These results demonstrate the capability of our OCT instrument to characterize in detail cardiac activity in genetic models for heart disease in Drosophila.