High-speed, high-resolution optical coherence microscopy combined with dual-channel fluorescence microscopy for small animal models imaging

Abstract

Purpose : Usage of small animal models has become a common practice in understanding pathological conditions. The purpose of this work is to develop a non-invasive, high-speed, and high-resolution optical coherence microscopy (OCM) combined with a dual-channel scanning fluorescence microscopy (FM) to enable longitudinal studies of the cornea in mouse models.

Methods : A high-resolution OCM was developed using a source (central wavelength at 850 nm and FWHM of 165 nm) that offers a theoretical axial resolution of ~1.5 µm (in tissue). A dual-detection channel FM system with two excitation peaks at 488 nm and 594 nm for green and red fluorophore variants was integrated with OCM. Beams from both systems were combined and focused onto the sample using a 10X objective, which yields a lateral resolution of ~2 µm. To evaluate this system, a conditional knockout mouse model to express tdTomato transgene that converts into green fluorescent proteins (GFP) on exposure to Cre recombinase was developed using the tet-O-Cre system. Rosa mT/mG reporter mice were crossed with the Keratocan rtTA/ tet-O-Cre transgenic mice to express the tdTomato gene in the corneal stroma. On treatment with doxycycline, part of the tdTomato transgene in the stroma gets converted to GFP over time. Both red and green fluorescence, as well as structural information of the stroma, were simultaneously imaged on a sacrificed mouse using the combined system.

Results : OCM achieved an axial resolution of ~2.4 µm in the cornea, which was slightly lower than the theoretical value due to dispersion. A 2.2 µm lateral resolution was demonstrated with the combined system. Reflectance and fluorescence were registered simultaneously with the speed of 250 kHz. Fig.1 shows a 1.3 × 1.3 mm² image of the mouse cornea. A 0.3 × 0.3 mm² field of view of the different layers of the cornea is shown in Fig.2, highlighting the epithelial cells, the keratocytes, and endothelial cells.

Conclusions : A combined system integrating a high-resolution OCM and a dual-channel FM was developed and evaluated in imaging mouse cornea. Future work will focus on improving the depth sectioning of FM to enable 3D reflectance and fluorescence in vivo imaging of the mouse cornea.

This is a 2021 ARVO Annual Meeting abstract.

 

Fig.1 Reflectance and fluorescence images of mouse cornea using OCM+FM.

 

Fig.2 Small field of view enface images of mouse cornea using OCM only.