High Resolution Macroscopic (HRMac) Imaging of the Mouse Meibomian Gland

Abstract

 

Purpose:

 

Recent studies suggest that the mouse meibomian gland (MG) develops atrophic changes with age similar to that of human. A better understanding of the structural changes that occur with age may provide important insights into age related MG dysfunction. The purpose of this study was to develop segmentation algorithms for the 3-dimensional reconstruction of the mouse MG that could then be used to volumetrically measure changes in gland structure.

 

Methods:

 

Non-linear optical imaging combined with computer assisted array tomography was used to 3-dimensionally reconstruct the mouse MG. Mouse eyelids were fixed in 4% paraformaldehyde, embedded in LR White and serially sectioned. Sections were then scanned using a 20x objective and a series of tiled images (1.4 mm by 1.4 mm) with a resolution of 0.44 µm lateral and 3 µm axial were collected using a Zeiss 510 Meta LSCM and femtosecond laser to generate second harmonic (SHG) signals from collagen and two-photon excited fluorescence (TPEF) signals from cells. Image tiles were then digitally aligned, and the SHG signal used to outline and generate an MG mask used to make surface renderings and extract MG TPEF signals with Amira software.

 

Results:

 

SHG was useful in clearly delineating the surface morphology (Fig. 1A) and extract the cellular TPEF signal (Fig. 1B) from MG.

 

Conclusions:

 

Using HRMac imaging, 3-dimensional reconstructions of the mouse MG can be generated that can be used for measuring the volume of meibomian gland duct and acini.  

 

Keywords: cornea: tears/tear film/dry eye • aging • lipids