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Improved tracking of corneal immune cell dynamics using confocal microscopy. | LitMetric

AI Article Synopsis

  • Confocal microscopy (IVCM) is an effective, non-invasive imaging technique for studying the cornea, but it faces challenges due to eye motion, leading to noise and limited visualization.
  • To improve image quality, a new data acquisition protocol and a free, open-source Matlab software package have been developed that register and process IVCM videos, enhancing contrast and resolution.
  • The software has demonstrated a high success rate in tracking immune cells in the corneal epithelium and stroma, supporting both cross-sectional and longitudinal studies in 68 people, with successful video registration rates of 97% for the epithelium and 93% for the stroma.

Article Abstract

confocal microscopy (IVCM) is a widely used technique for imaging the cornea of the eye with a confocal scanning light ophthalmoscope. Cellular resolution and high contrast are achieved without invasive procedures, suiting the study of living humans. However, acquiring useful image data can be challenging due to the incessant motion of the eye, such that images are typically limited by noise and a restricted field of view. These factors affect the degree to which the same cells can be identified and tracked over time. To redress these shortcomings, here we present a data acquisition protocol together with the details of a free, open-source software package written in Matlab. The software package automatically registers and processes IVCM videos to significantly improve contrast, resolution, and field of view. The software also registers scans acquired at progressive time intervals from the same tissue region, producing a time-lapsed video to facilitate visualization and quantification of individual cell dynamics (e.g., motility and dendrite probing). With minimal user intervention, to date, this protocol has been employed to both cross-sectionally and longitudinally assess the dynamics of immune cells in the human corneal epithelium and stroma, using a technique termed functional in vivo confocal microscopy (Fun-IVCM) in 68 eyes from 68 participants. Using the custom software, registration of 'sequence scan' data was successful in 97% of videos acquired from the corneal epithelium and 93% for the corneal stroma. Creation of time-lapsed videos, in which the averages from single videos were registered across time points, was successful in 93% of image series for the epithelium and 75% of image series for the stroma. The reduced success rate for the stroma occurred due to practical difficulties in finding the same tissue between time points, rather than due to errors in image registration. We also present preliminary results showing that the protocol is well suited to cellular imaging in the retina with adaptive optics scanning laser ophthalmoscopy (AOSLO). Overall, the approach described here substantially improves the efficiency and consistency of time-lapsed video creation to enable non-invasive study of cell dynamics across diverse tissues in the living eye.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC11563322PMC
http://dx.doi.org/10.1364/BOE.536553DOI Listing

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