Bright-Field Imaging and Optical Coherence Tomography of the Mouse Posterior Eye

Abstract

Noninvasive live imaging has been used extensively for ocular phenotyping in mouse vision research. Bright-field imaging and optical coherence tomography (OCT) are two methods that are particularly useful for assessing the posterior mouse eye (fundus), including the retina, retinal pigment epithelium, and choroid, and are widely applied due to the commercial availability of sophisticated instruments and software. Here, we provide a guide to using these approaches with an emphasis on post-acquisition image processing using Fiji, a bundled version of the Java-based public domain software ImageJ. A bright-field fundus imaging protocol is described for acquisition of multi-frame videos, followed by image registration to reduce motion artifacts, averaging to reduce noise, shading correction to compensate for uneven illumination, filtering to improve image detail, and rotation to adjust orientation. An OCT imaging protocol is described for acquiring replicate volume scans, with subsequent registration and averaging to yield three-dimensional datasets that show reduced motion artifacts and enhanced detail. The Fiji algorithms used in these protocols are designed for batch processing and are freely available. The image acquisition and processing approaches described here may facilitate quantitative phenotyping of the mouse eye in drug discovery, mutagenesis screening, and the functional cataloging of mouse genes by individual laboratories and large-scale projects, such as the Knockout Mouse Phenotyping Project and International Mouse Phenotyping Consortium.

Mouse Models of Inherited Retinal Degeneration with Photoreceptor Cell Loss

“…Fundus photodocumentation for TVRM mutants and C57BL/6J control mice was performed using a Micron III or IV retinal camera (Phoenix Laboratories, Inc., Pleasanton, CA, USA) as described [37], except that 1% cyclopentolate or 1% atropine was used as a dilating agent, and in some cases, mice were anesthetized with isoflurane. OCT imaging to assess retinal layer thickness in Nmnat1 tvrm113 , Ctnna1 Tvrm5 , and C57BL/6J control mice was performed using a Bioptigen ultrahigh-resolution (UHR) Envisu R2210 spectral domain OCT (SDOCT) imaging system for volume scanning as described [37,38] with ketamine/xylazine (1.6 mL ketamine (100 mg/mL), 1.6 mL xylazine (20 mg/mL), and 6.8 mL sodium chloride (0.9% w/v)) as an anesthetic. A representative B-scan through the optic nerve head was derived from the OCT volume dataset.…”

Using Vascular Landmarks to Orient 3D Optical Coherence Tomography Images of the Mouse Eye

“…However, to make use of additional functionality that is unavailable in the manufacturer’s software, researchers may choose to export image data for processing and analysis with third-party software. For example, the freeware Fiji (Schindelin et al, 2012), a bundled version of ImageJ2 (Schindelin, Rueden, Hiner, & Eliceiri, 2015), may be used to align, average and analyze 3D OCT datasets (Krebs et al, 2016). This program provides a large suite of image processing algorithms as well as the possibility of macro and plugin development tailored for image data analysis.…”

“…Run OCT Volume Averager on a multiple OCT files acquired as described previously (Krebs et al, 2016). …”

“…In one corrective approach, we attempted to account for orientation in brightfield fundus images by using an image of the external eye. Fundus images were rotated by an angle opposite to the angle of rotation of the edge of the nictitating membrane relative to the center of the pupil (Krebs et al, 2016; (Low et al, 2014), but this approach remains to be validated. We are unaware of methods to orient noninvasive images of the eye acquired from other commonly used non-primate research animals.…”

“…Recently, we presented images identifying a portion of the LPCAs in 3D OCT datasets of the mouse posterior eye centered on the optic nerve head (Krebs, Xiao, Sheppard, Hicks, & Nishina, 2016). We proposed that these vessels might be useful as rotational landmarks.…”