Motile cilia lining the lumen of the Fallopian tube (oviduct) are known to play an essential role in reproduction; however, how their specific roles are not understood. Here, we present a novel dynamic OCT method for imaging of waves propagating along the epithelial surfaces created by the beating cilia (metachronal waves). The method is based on spatiotemporal mapping of the phase of ciliary beat calculated based on intensity fluctuations in OCT images. This method was applied in vivo to visualize and quantify cilia metachronal waves within the mouse fallopian tube in vivo, volumetrically, through the tissue layers.
Cilia beat frequency (CBF) is an essential measure of fallopian tube function. In the present study, we adapted a previously developed functional optical coherence tomography (OCT) method, to spatially map and quantify CBF in one human sample ex vivo. Time lapsed image sets at different locations were acquired using OCT (n=8) and 50x magnification brightfield (BF) microscopy (n=2) as ground truth. A sliding window Fourier analysis was performed on OCT and BF image sets to quantify CBF on a pixel-by-pixel basis. Parameters were optimized to maximize contrast of cilia, and were uniformly applied for all OCT image sets. CBF color maps were created and were qualitatively compared to unprocessed OCT intensity image sets to evaluate the spatial mapping accuracy of CBF values. Line plots of amplitude vs. frequency at 1 second intervals, and pixel peak frequency histograms of whole image sets, were created to visualize the dominant CBFs. An analysis of variance was used to compare CBFs as measured with OCT and BF microscopy. Our results revealed qualitatively accurate spatial mapping of non-zero CBF values to pixels in ciliated areas, which were visibly appreciable on unprocessed intensity image sets. There was no significant difference between the dominant CBFs as measured with OCT and ground truth BF microscopy (3.2 ± 1.6Hz, 3.2 ± 0, p=0.97). Bulk sample movement was a significant source of temporal variability and amplified high-frequency noise.
Motile cilia are hair-like microtubule-based organelles covering epithelial surfaces of multiple organ systems, including lungs, airways, kidneys, and fallopian tubes. Genetic alterations affecting cilia structure and motility are associated with ciliopathies affecting the physiology of multiple organs, including reduced fertility and a higher incidence of ectopic pregnancies. Motile cilia beat periodically to propel fluids, mucus, and cells along the epithelial surfaces. The ciliary beating is coordinated between neighboring cells. Due to a phase shift, the beat of cilia forms a wave propagating along the surface, which is called the metachronal wave. While the clinical importance of cilia and their coordinated function is well recognized, this function is very poorly studied due to the lack of cilia imaging methods. Previously, we established a functional OCT method for mapping cilia and cilia beat frequency within the mouse fallopian tube in vivo, volumetrically, through the tissue layers. That method was based on the analysis of periodic OCT intensity fluctuations in pixels corresponding to cilia. Building on that method, here we present the visualization and quantification of cilia metachronal wave, by spatio-temporal analysis of ciliary beat phase propagation. This study presented the first quantitative approach for measuring cilia metachronal waves through tissue layers. This method gives unique access to cilia function in the mammalian fallopian tube in vivo, as demonstrated here. It can also be directly adapted to investigating coordinated cilia behaviors in multiple organ systems toward better-managing pathologies associated with ciliopathies.
KEYWORDS: Optical coherence tomography, In vivo imaging, Visualization, Optical tracking, Speckle, Signal detection, In vitro testing, Imaging systems, Control systems, Microscopy
Fertilization is a critical reproductive step combining two gametes from parents, the egg and the sperm, to convey the genetic information to the next generation. This process is regulated in vivo by multiple factors influencing a selection of one sperm out of millions. However, due to the lack of suitable visualization approaches for in vivo fertilization processes, most of the current knowledge is derived from in vitro experiments, which cannot model the complexity of physiological states. The overall goal of this project is to capture the fertilization in vivo in a mouse model and to characterize the mechanisms regulating the dynamics of this process. Toward this goal, we recently established functional optical coherence tomography (OCT) integrated with intravital microscopy for live dynamic imaging of reproductive processes within the mouse fallopian tube. This method allowed for prolonged volumetric tracking of oocytes and embryos as well as individual spermatozoa at the site of fertilization based on their trajectories. However, visualizing the fertilization event remains a major challenge because the oocytes prior to fertilization are surrounded by a dense cloud of cumulus cells, forming the cumulus-oocyte-complex (COC), making direct sperm tracking through the optically dense cumulus matrix not feasible. This study presents the development of the speckle variance (SV) OCT analysis for volumetric tracking of sperm migration through the cumulus matrix toward the oocyte in vitro. Potentially, this method will be integrated with intravital OCT imaging to capture the process of mammalian fertilization in vivo.
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