KEYWORDS: Optical coherence tomography, Tissues, Image segmentation, Refractive index, Lung, Refraction, 3D modeling, In vivo imaging, Error analysis, 3D metrology
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.
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.
In vitro fertilization (IVF) procedures have resulted in the birth of over three million babies since 1978. Yet the
live birth rate in the United States was only 34% in 2005, with 32% of the successful pregnancies resulting in multiple
births. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability
that a single, healthy embryo was included. Current viability markers used for IVF, such as the cell number, symmetry,
size, and fragmentation, are analyzed qualitatively with differential interference contrast (DIC) microscopy. However,
this method is not ideal for quantitative measures beyond the 8-cell stage of development because the cells overlap and
obstruct the view within and below the cluster of cells. We have developed the phase-subtraction cell-counting method
that uses the combination of DIC and optical quadrature microscopy (OQM) to count the number of cells accurately in
live mouse embryos beyond the 8-cell stage. We have also created a preliminary analysis to measure the cell symmetry,
size, and fragmentation quantitatively by analyzing the relative dry mass from the OQM image in conjunction with the
phase-subtraction count. In this paper, we will discuss the characterization of OQM with respect to measuring the phase
accurately for spherical samples that are much larger than the depth of field. Once fully characterized and verified with
human embryos, this methodology could provide the means for a more accurate method to score embryo viability.
Several quantitative phase imaging techniques, such as digital holography, Hilbert-phase microscopy, and phase-shifting
interferometry have applications in biological and medical imaging. Quantitative phase imaging measures
the changes in the wavefront of the incident light due to refractive index variations throughout a 3-D specimen. We
have developed a multimodal microscope which combines optical quadrature microscopy (OQM) and a Shack-
Hartmann wavefront sensor for applications in biological imaging. OQM is an interferometric imaging modality
that noninvasively measures the amplitude and phase of a signal beam that travels through a transparent specimen.
The phase is obtained from interferograms with four different delayed reference wavefronts. The phase is then
transformed into a quantitative image of optical path length difference. The Shack-Hartmann wavefront sensor
measures the gradient of the wavefront at various points across a beam. A microlens array focuses the local
wavefront onto a specific region of the CCD camera. The intensity is given by the maximum amplitude in the
region and the phase is determined based on the exact pixel position within the region.
We compare the amplitude and quantitative phase information of poly-methyl-meth-acrylate (PMMA) beads in oil
and one-cell and two-cell mouse embryos with micrometer resolution using OQM and the Shack-Hartmann. Each
pixel in OQM provides a phase measurement, whereas multiple pixels are used in Shack-Hartmann to determine the
tilt. Therefore, the simple Shack-Hartmann system is limited by its resolution and field-of-view. Real-time imaging
in Shack-Hartmann requires spatial averaging which smoothes the edges of the PMMA beads. The OQM has a
greater field-of-view with good resolution; however, it is a complex system requiring multiple optical components
and four cameras which may introduce additional artifacts in processing quantitative images. The OQM and Shack-
Hartmann has certain advantages depending on the application. A combination of these two systems may provide
improved quantitative phase information than either one alone.cHJl
Confocal microscopes achieve high spatial resolution by focusing both a light source and a detector to a single
point with an objective having a high numerical aperture. In order to produce an image, it is then necessary to
scan this common focal spot through the specimen, and it is often important to image the full field of view in a
short time. In order to avoid vignetting, the scanning must be done in or near the pupil of the optical system.
For some fast scanners, this requires the use of multiple relay telescopes to create multiple pupils. Galvanometric
scanners impose a practical limit on the scan speed because of the angular accelerations involved in reversing
direction. Rotating polygons are often used to achieve greater speed. For a scanner consisting of a rotating
polygon and a galvanometric scanner, two relay telescopes are normally used.
We have developed a dual-wedge scanner which has the potential to perform the scan in a configuration which
is very short in the axial direction, thereby eliminating much of the complexity of current high-speed scanners.
We have demonstrated a prototype of the scanner in a reflectance confocal microscope. Transverse and axial
resolution are comparable to those of other scanning systems.
The selection of rotation speeds for the wedges is important to ensuring full coverage of the field of view in
a reasonable time. Various tradeoffs on these parameters will be discussed. The beam behavior in the pupil
will be discussed. Resolution limits and aberrations will be shown from ray-tracing analysis, and compared to
experimental results.
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.
Optical quadrature microscopy (OQM) has been shown to provide the optical path difference through a mouse embryo, and has led to a novel method to count the total number of cells further into development than current non-toxic imaging techniques used in the clinic. The cell counting method has the potential to provide an additional quantitative viability marker for blastocyst transfer during in vitro fertilization. OQM uses a 633 nm laser within a modified Mach-Zehnder interferometer configuration to measure the amplitude and phase of the signal beam that travels through the embryo. Four cameras preceded by multiple beamsplitters record the four interferograms that are used within a reconstruction algorithm to produce an image of the complex electric field amplitude. Here we present a model for the electric field through the primary optical components in the imaging configuration and the reconstruction algorithm to calculate the signal to noise ratio when imaging mouse embryos. The model includes magnitude and phase errors in the individual reference and sample paths, fixed pattern noise, and noise within the laser and detectors. This analysis provides the foundation for determining the imaging limitations of OQM and the basis to optimize the cell counting method in order to introduce additional quantitative viability markers.
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.
Confocal reflectance microscopy has been shown to provide optical sectioning and resolution sufficient to provide useful
information about cellular structure in vivo. However, existing instruments are large and expensive, because of the need
for fast, two-dimensional scanning in the pupil, and the associated relay optics. A more compact scanning system could
lead to an affordable handheld instrument for in vivo imaging. Several approaches are being considered to minimize
instrument size with different advantages and disadvantages. Here we report one approach that incorporates a dualwedge
scanner within a point-scanning configuration. The dual-wedge scanner could reduce the cost and complexity of
the confocal reflectance microscope while retaining the resolution and optical sectioning abilities of current pointscanning
instruments. The scanner is implemented by replacing the scanning mirrors and the relay telescope between
them with two optical prisms that are rotated about the optical axis. This scanning configuration produces a spiral scan
if the prisms are rotated in the same direction, or a rosette scan if the prisms are rotated in opposite directions.
Preliminary experimental results with the microscope show a lateral resolution on the order of 1 - 2 micrometers and onaxis
optical sectioning on the order of 3 - 4 micrometers.
Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight-cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 13-25 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy. Optical Quadrature Microscopy is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal model cell of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is analyzed to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 5 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.
The Multifunctional Staring Mode Microscope was developed to permit three modes of imaging for cell counting in mouse embryos: Optical Quadrature, Differential Interference Contrast (DIC), and Fluorescence Imaging. The Optical Quadrature Microscope, consisting of a modified Mach-Zender Interferometer, uses a 632.8 nm laser to measure the amplitude and phase of the signal beam that travels through the embryo. Four cameras, preceded by multiple beamsplitters, are used to read the four interferograms, which are then combined to produce an image of the complex electric field amplitude. The phase of the complex amplitude is then unwrapped using a 2-D phase unwrap algorithm and images of optical path length are produced. To combine the additional modes of DIC and Fluorescence Imaging with the Optical Quadrature Microscope, a 632.8 nm narrow bandpass beamsplitter was placed at the output of the microscope. This allows the laser light to continue through the Mach-Zender while all other wavelengths are reflected at 90 degrees to another camera. This was effective in combining the three modes as the fluorescence wavelength for the Hoechst stain is well below the bandpass window of the beamsplitter. Both live and fixed samples have been successfully imaged in all three modes. Accuracy in cell counting was achieved by using the DIC image for detecting cell boundaries and the Optical Quadrature image for phase mapping to determine where cells overlap. The final results were verified by Hoechst fluorescence imaging to count the individual nuclei. Algorithms are currently being refined so larger cell counts can be done more efficiently.
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