Maria Gaertner

Optical coherence tomography in biomedical research.

Optical coherence tomography (OCT) is a noninvasive, high-resolution, interferometric imaging modality using near-infrared light to acquire cross-sections and three-dimensional images of the subsurface microstructure of biological specimens. Because of rapid improvement of the acquisition speed and axial resolution of OCT over recent years, OCT is becoming increasingly attractive for applications in biomedical research. Therefore, OCT is no longer used solely for structural investigations of biological samples but also for functional examination, making it potentially useful in bioanalytical science. The combination of in vivo structural and functional findings makes it possible to obtain thorough knowledge on basic physiological and pathological processes. Advanced applications, for example, optical biopsy in visceral cavities, have been enabled by combining OCT with established imaging modalities. This report gives an outline of the state of the art and novel trends of innovative OCT approaches in biomedical research in which the main focus is on applications in fundamental research and pre-clinical utilization.

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.

Three-dimensional simultaneous optical coherence tomography and confocal fluorescence microscopy for investigation of lung tissue

Abstract. Although several strategies exist for a minimal-invasive treatment of patients with lung failure, the mortality rate of acute respiratory distress syndrome still reaches 30% at minimum. This striking number indicates the necessity of understanding lung dynamics on an alveolar level. To investigate the dynamical behavior on a microscale, we used three-dimensional geometrical and functional imaging to observe tissue parameters including alveolar size and length of embedded elastic fibers during ventilation. We established a combined optical coherence tomography (OCT) and confocal fluorescence microscopy system that is able to monitor the distension of alveolar tissue and elastin fibers simultaneously within three dimensions. The OCT system can laterally resolve a 4.9 μm line pair feature and has an approximately 11 μm full-width-half-maximum axial resolution in air. confocal fluorescence microscopy visualizes molecular properties of the tissue with a resolution of 0.75 μm (laterally), and 5.9 μm (axially) via fluorescence detection of the dye sulforhodamine B specifically binding to elastin. For system evaluation, we used a mouse model in situ to perform lung distension by application of different constant pressure values within the physiological regime. Our method enables the investigation of alveolar dynamics by helping to reveal basic processes emerging during artificial ventilation and breathing.

Total liquid ventilation: a new approach to improve 3D OCT image quality of alveolar structures in lung tissue

Little is known about mechanical processes of alveolar tissue during mechanical ventilation. Optical coherence tomography (OCT) as a three-dimensional and high-resolution imaging modality can be used to visualize subpleural alveoli during artificial ventilation. The quality of OCT images can be increased by matching the refractive index inside the alveoli to the one of tissue via liquid-filling. Thereby, scattering loss can be decreased and higher penetration depth and tissue contrast can be achieved. We show the liquid-filling of alveolar structures verified by optical coherence tomography and intravital microscopy (IVM) and the advantages of index matching for OCT imaging of subpleural alveoli in a mouse model using a custom-made liquid ventilator.

Imaging of nanoparticle-labeled stem cells using magnetomotive optical coherence tomography, laser speckle reflectometry, and light microscopy

Abstract. Cell transplantation and stem cell therapy are promising approaches for regenerative medicine and are of interest to researchers and clinicians worldwide. However, currently, no imaging technique that allows three-dimensional in vivo inspection of therapeutically administered cells in host tissues is available. Therefore, we investigate magnetomotive optical coherence tomography (MM-OCT) of cells labeled with magnetic particles as a potential noninvasive cell tracking method. We develop magnetomotive imaging of mesenchymal stem cells for future cell therapy monitoring. Cells were labeled with fluorescent iron oxide nanoparticles, embedded in tissue-mimicking agar scaffolds, and imaged using a microscope setup with an integrated MM-OCT probe. Magnetic particle-induced motion in response to a pulsed magnetic field of 0.2 T was successfully detected by OCT speckle variance analysis, and cross-sectional and volumetric OCT scans with highlighted labeled cells were obtained. In parallel, fluorescence microscopy and laser speckle reflectometry were applied as two-dimensional reference modalities to image particle distribution and magnetically induced motion inside the sample, respectively. All three optical imaging modalities were in good agreement with each other. Thus, magnetomotive imaging using iron oxide nanoparticles as cellular contrast agents is a potential technique for enhanced visualization of selected cells in OCT.

Quantitative investigation of alveolar structures with OCT using total liquid ventilation during mechanical ventilation

To develop new treatment possibilities for patients with severe lung diseases it is crucial to understand the lung function on an alveolar level. Optical coherence tomography (OCT) in combination with intravital microscopy (IVM) are used for imaging subpleural alveoli in animal models to gain information about dynamic and morphological changes of lung tissue during mechanical ventilation. The image content suitable for further analysis is influenced by image artifacts caused by scattering, refraction, reflection, and absorbance. Because the refractive index varies with each air-tissue interface in lung tissue, these effects decrease OCT image quality exceedingly. The quality of OCT images can be increased when the refractive index inside the alveoli is matched to the one of tissue via liquid-filling. Thereby, scattering loss can be decreased and higher penetration depth and tissue contrast can be achieved. To use the advantages of liquid-filling for in vivo imaging of small rodent lungs, a suitable breathing fluid (perfluorodecalin) and a special liquid respirator are necessary. Here we show the effect of liquid-filling on OCT and IVM image quality of subpleural alveoli in a mouse model.

Optical coherence tomography and confocal fluorescence microscopy as a combined method for studying morphological changes in lung dynamics

Acute lung injury (ALI) is a severe pulmonary disease leading to hypoxemia accompanied by a reduced compliance and partial edema of the lung. Most of the patients have to be ventilated to compensate for the lack of oxygen. The treatment is strongly connected with ventilator induced lung injury (VILI), which is believed to introduce further stress to the lung and changes in its elastic performance. A thorough understanding of the organs micro-structure is crucial to gain more insight into the course of the disease. Due to backscattering of near-infrared light, detailed description of lung morphology can be obtained using optical coherence tomography (OCT), a non-invasive, non-contact, high resolution and fast three-dimensional imaging technique. One of its drawbacks lies in the non-specificity of light distribution in relation to defined substances, like elastic biomolecules. Using fluorescence detection, these chemical components can be visualized by introducing specifically binding fluorophores. This study presents a combined setup for studying alveolar compliance depending on volume changes and elastic fiber distributions. Simultaneously acquired OCT and confocal fluorescence images allow an entire view into morphological rearrangements during ventilation for an ex vivo mouse model using continuous pulmonary airway pressure at different values.

Investigation of alveolar tissue deformations using OCT combined with fluorescence microscopy

In critical care medicine, artificial ventilation is a life saving tool providing sufficient blood oxygenation to patients suffering from respiratory failure. Essential for their survival is the use of protective ventilation strategies to prevent further lung damage due to ventilator induced lung injury (VILI). Since there is only little known about implications of lung tissue overdistension on the alveolar level, especially in the case of diseased lungs, this research deals with the investigation of lung tissue deformation on a microscale. A combined setup utilizing optical coherence tomography (OCT) and confocal fluorescence microscopy, is used to study the elastic behavior of the alveolar tissue. Three-dimensional geometrical information with voxel sizes of 6 μm × 6 μm × 11 μm (in air) is provided by OCT, structural information about localization of elastin fibers is elucidated via confocal fluorescence microscopy with a lateral resolution of around 1 μm. Imaging depths of 90 μm for OCT and 20 μm for confocal fluorescence microscopy were obtained. Dynamic studies of subpleural tissue were carried out on the basis of an in vivo mouse model post mortem, mimicking the physiological environment of an intact thorax and facilitating a window for the application of optical methods. Morphological changes were recorded by applying constant positive airway pressures of different values. With this, alveolar volume changes could clearly be recognized and quantified to form a compliance value of 3.5 • 10-6(see manuscript). The distribution of elastin fibers was detected and will be subject to further elasticity analysis.

Optical coherence tomography for imaging of subpleural alveolar structure using a Fourier domain mode locked laser

Optical coherence tomography (OCT) is a noninvasive imaging modality generating cross sectional and volumetric images of translucent samples. In Fourier domain OCT (FD OCT), the depth profile is calculated by a fast Fourier transformation of the interference spectrum, providing speed and SNR advantage and thus making FD OCT well suitable in biomedical applications. The interference spectrum can be acquired spectrally resolved in spectral domain OCT or time-resolved in optical frequency domain imaging (OFDI). Since OCT images still suffer from motion artifacts, especially under in vivo conditions, increased depth scan rates are required. Therefor, the principle of Fourier domain mode locking has been presented by R. Huber et al. circumventing the speed limitations of conventional FD OCT systems. In FDML lasers, a long single mode fiber is inserted in the ring resonator of the laser resulting in an optical round trip time of a few microseconds. Sweeping the wavelength synchronously by a tunable Fabry-Perot filter can provide wavelength sweeps with repetition rates up to a few MHz used for OFDI. Imaging of subpleural lung tissue for investigation of lung dynamics and its elastic properties is a further biomedical application demanding high-speed OCT imaging techniques. For the first time, the visualization of subpleural alveolar structures of a rabbit lung is presented by the use of an FDML-based OCT system enabling repetition rates of 49.5 kHz and 122.6 kHz, respectively.

Combining Optical Coherence Tomography with Fluorescence Microscopy: A closer look into tissue

Optical coherence tomography (OCT) is a technique, capable of high resolution and non-invasive 3D imaging in vivo by detection of backscattered light from cellular and sub cellular structures. Due to visualization of micrometer sized tissue constituents and high penetration depths of up to 2 mm, it is already well established in medical fields like ophthalmology and dermatology. Laser scanning confocal microscopy (LSCM), on the contrary, gives further information on structural tissue components stained with suitable dyes. In combination, these two methods yield three dimensional and high resolution data providing geometrical and structural details of tissue. In this study, we present simultaneous OCT and LSCM image acquisition resulting in a lateral resolution of better than 6.2 μm for OCT and 0.8 μm for LSCM, respectively. The axial resolution of the OCT amounts to 8 μm. Two laser lines, 488 nm and 561 nm, are combined to provide fluorescence excitation of green and red dyes. By using a long working distance objective, it is possible to perform experiments on bulky specimens like isolated organs or animal models in vivo. First studies indicate the ability to identify strains of elastic fibers within lung tissue in combination with the three dimensional morphology of the lung.