Four-dimensional visualization of subpleural alveolar dynamics in vivo during uninterrupted mechanical ventilation of living swine.

Pulmonary alveoli have been studied for many years, yet no unifying hypothesis exists for their dynamic mechanics during respiration due to their miniature size (100-300 µm dimater in humans) and constant motion, which prevent standard imaging techniques from visualizing four-dimensional dynamics of individual alveoli in vivo. Here we report a new platform to image the first layer of air-filled subpleural alveoli through the use of a lightweight optical frequency domain imaging (OFDI) probe that can be placed upon the pleura to move with the lung over the complete range of respiratory motion. This device enables in-vivo acquisition of four-dimensional microscopic images of alveolar airspaces (alveoli and ducts), within the same field of view, during continuous ventilation without restricting the motion or modifying the structure of the alveoli. Results from an exploratory study including three live swine suggest that subpleural alveolar air spaces are best fit with a uniform expansion (r (2) = 0.98) over a recruitment model (r (2) = 0.72). Simultaneously, however, the percentage change in volume shows heterogeneous alveolar expansion within just a 1 mm x 1 mm field of view. These results signify the importance of four-dimensional imaging tools, such as the device presented here. Quantification of the dynamic response of the lung during ventilation may help create more accurate modeling techniques and move toward a more complete understanding of alveolar mechanics.

Validation of two-dimensional and three-dimensional measurements of subpleural alveolar size parameters by optical coherence tomography.

Optical coherence tomography (OCT) has been increasingly used for imaging pulmonary alveoli. Only a few studies, however, have quantified individual alveolar areas, and the validity of alveolar volumes represented within OCT images has not been shown. To validate quantitative measurements of alveoli from OCT images, we compared the cross-sectional area, perimeter, volume, and surface area of matched subpleural alveoli from microcomputed tomography (micro-CT) and OCT images of fixed air-filled swine samples. The relative change in size between different alveoli was extremely well correlated (r>0.9, P<0.0001), but OCT images underestimated absolute sizes compared to micro-CT by 27% (area), 7% (perimeter), 46% (volume), and 25% (surface area) on average. We hypothesized that the differences resulted from refraction at the tissue-air interfaces and developed a ray-tracing model that approximates the reconstructed alveolar size within OCT images. Using this model and OCT measurements of the refractive index for lung tissue (1.41 for fresh, 1.53 for fixed), we derived equations to obtain absolute size measurements of superellipse and circular alveoli with the use of predictive correction factors. These methods and results should enable the quantification of alveolar sizes from OCT images in vivo.

Differentiation of early from advanced coronary atherosclerotic lesions: systematic comparison of CT, intravascular US, and optical frequency domain imaging with histopathologic examination in ex vivo human hearts.

PURPOSE: To establish an ex vivo experimental setup for imaging coronary atherosclerosis with coronary computed tomographic (CT) angiography, intravascular ultrasonography (US), and optical frequency domain imaging (OFDI) and to investigate their ability to help differentiate early from advanced coronary plaques. MATERIALS AND METHODS: All procedures were performed in accordance with local and federal regulations and the Declaration of Helsinki. Approval of the local Ethics Committee was obtained. Overall, 379 histologic cuts from nine coronary arteries from three donor hearts were acquired, coregistered among modalities, and assessed for the presence and composition of atherosclerotic plaque. To assess the discriminatory capacity of the different modalities in the detection of advanced lesions, c statistic analysis was used. Interobserver agreement was assessed with the Cohen κ statistic. RESULTS: Cross sections without plaque at coronary CT angiography and with fibrous plaque at OFDI almost never showed advanced lesions at histopathologic examination (odds ratio [OR]: 0.02 and 0.06, respectively; both P<.0001), while mixed plaque at coronary CT angiography, calcified plaque at intravascular US, and lipid-rich plaque at OFDI were associated with advanced lesions (OR: 2.49, P=.0003; OR: 2.60, P=.002; and OR: 31.2, P<.0001, respectively). OFDI had higher accuracy for discriminating early from advanced lesions than intravascular US and coronary CT angiography (area under the receiver operating characteristic curve: 0.858 [95% confidence interval {CI}: 0.802, 0.913], 0.631 [95% CI: 0.554, 0.709], and 0.679 [95% CI: 0.618, 0.740]; respectively, P<.0001). Interobserver agreement was excellent for OFDI and coronary CT angiography (κ=0.87 and 0.85, respectively) and was good for intravascular US (κ=0.66). CONCLUSION: Systematic and standardized comparison between invasive and noninvasive modalities for coronary plaque characterization in ex vivo specimens demonstrated that coronary CT angiography and intravascular US are reasonably associated with plaque composition and lesion grading according to histopathologic findings, while OFDI was strongly associated. These data may help to develop initial concepts of sequential imaging strategies to identify patients with advanced coronary plaques.

Evaluation of optical reflectance techniques for imaging of alveolar structure.

Three-dimensional (3-D) visualization of the fine structures within the lung parenchyma could advance our understanding of alveolar physiology and pathophysiology. Current knowledge has been primarily based on histology, but it is a destructive two-dimensional (2-D) technique that is limited by tissue processing artifacts. Micro-CT provides high-resolution three-dimensional (3-D) imaging within a limited sample size, but is not applicable to intact lungs from larger animals or humans. Optical reflectance techniques offer the promise to visualize alveolar regions of the large animal or human lung with sub-cellular resolution in three dimensions. Here, we present the capabilities of three optical reflectance techniques, namely optical frequency domain imaging, spectrally encoded confocal microscopy, and full field optical coherence microscopy, to visualize both gross architecture as well as cellular detail in fixed, phosphate buffered saline-immersed rat lung tissue. Images from all techniques were correlated to each other and then to corresponding histology. Spatial and temporal resolution, imaging depth, and suitability for in vivo probe development were compared to highlight the merits and limitations of each technology for studying respiratory physiology at the alveolar level.

Computational signal-to-noise ratio analysis for optical quadrature microscopy.

Optical quadrature microscopy (OQM) was invented in 1997 to reconstruct a full-field image of quantitative phase, and has been used to count the number of cells in live mouse embryos. Here we present a thorough SNR analysis that incorporates noise terms for fluctuations in the laser, aberrations within the individual paths of the Mach-Zehnder interferometer, and imperfections within the beamsplitters and CCD cameras to create a model for the resultant phase measurements. The current RMS error of the OQM phase images has been calculated to be 0.08 radians from substituting images from the instrumentation into the model.

Phase-subtraction cell-counting method for live mouse embryos beyond the eight-cell stage.

Since 1978 in vitro fertilization (IVF) procedures have resulted in the birth of over 3 million babies. Yet in 2005, IVF procedures had a live birth rate of only 34%, with 32% of these births resulting in multiple pregnancies. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability that a single, healthy embryo was included. The predominantly accepted noninvasive viability markers for embryos created by IVF are (1) number of cells at specific time points during development and (2) overall morphology of the embryo. Currently, it is difficult to count the number of cells beyond the eight-cell stage noninvasively. We report a nontoxic cell-counting method capable of counting cell numbers ranging from 8 to 26 in live mouse embryos. This method is derived from the fusion of differential interference contrast and optical quadrature microscopy and is verified by epifluorescence images of Hoechst-stained nuclei. The phase-subtraction cell-counting method is the first accurate, nontoxic technique to count cells through the morula stage in mouse embryos and may enhance the use of cell number as a viability marker if adopted for use with human embryos in the IVF clinic.

Multimodal optical microscope for detecting viability of mouse embryos in vitro.

We present a multimodal optical microscope that incorporates six imaging modalities on one common platform. The imaging modalities include three staring modes, optical quadrature microscopy (OQM), differential interference contrast (DIC) microscopy, and epi-fluorescence microscopy, and three scanning modes, confocal reflectance microscopy (CRM), confocal fluorescence microscopy (CFM), and two-photon microscopy (2PM). OQM reconstructs the amplitude and phase of an optically transparent specimen within a modified Mach-Zehnder configuration. DIC microscopy images the phase gradient along a specified direction of an optically transparent specimen. CRM detects index of refraction changes that modulate backscatter. Epi-fluorescence microscopy, CFM, and 2PM detect endogenous and exogenous fluorophores within a specimen. The scanning modes are inherently capable of producing three-dimensional (3-D) images due to optical sectioning and localized probing. Illumination and imaging are performed coaxially with minimal changes of optical components between modes. Multimodal images of embryos are shown to demonstrate the microscope's imaging capabilities.

Dual-wedge scanning confocal reflectance microscope.

A confocal reflectance microscope has been developed that incorporates a dual-wedge scanner to reduce the size of the device relative to current raster scanning instruments. The scanner is implemented with two prisms that are rotated about the optical axis. Spiral and rosette scans are performed by rotating the prisms in the same or opposite directions, respectively. Experimental measurements show an on-axis lateral resolution of 1.6 microm and optical sectioning of 4.7 microm, which compares with a diffraction-limited resolution of 0.8 and 1.9 microm, respectively.

Determination of the number of cells in preimplantation embryos by using noninvasive optical quadrature microscopy in conjunction with differential interference contrast microscopy.

The number of cells in a preimplantation embryo is directly correlated to the health and viability of the embryo. There are currently no methods to count the number of cells in late-stage preimplantation embryos noninvasively. We assessed the ability of optical quadrature microscopy (OQM) to count the number of cells in mouse preimplantation embryos noninvasively. First, to test for possible light toxicity, we exposed two-cell mouse embryos to OQM and differential interference contrast (DIC) microscopy and assessed their ability to develop to the blastocyst stage. We found no inhibition of development from either mode of microscopy for up to 2 h of light exposure. We also imaged eight-cell to morula stage mouse preimplantation embryos by OQM nd developed two methods for counting the number of cells. The contour signature method (CSM) used OQM images alone and the phase subtraction method (PSM) used both OQM and DIC images. We compared both methods to standard cell counting techniques and found that the PSM was superior to all other noninvasive cell counting methods. Our work on mouse embryos should be applicable to human embryos. The ability to correctly count the number of cells in human preimplantation embryos could lead to the transfer of fewer embryos in in vitro fertilization (IVF) clinics and consequently a lower rate of high-risk multiple-infant births.