VU Biomolecular Multimodal Imaging Center (BIOMIC) Eye characterization pipeline

Collaborating with the Advancing Sight Network (500 Robert Jemison Rd Birmingham, AL 35209), our team identifies and preserves human donor ocular tissue using the following protocols.

Donor Tissue Acceptance Criteria:

1. Caucasian or African American (AA)

2. ≥18-70 years of age

3. Acceptable: sepsis

4. Acceptable: long or short optic nerve stump

Exclusion Criteria:

1. Diabetes

2. Ventilator ≥5 days

3. Cataract surgery; glaucoma shunt; intravitreal injections; cornea donor, cornea with infiltrates

4. Conditions discovered through the Interview that can affect retina or choroid (i.e., AMD, macular edema, macular hole, retinitis pigmentosa; neurologic disease Parkinson, any dementia)

5. Surgeries affecting retina, discovered by Interview: vitrectomy, pan-retinal coagulation, scleral buckle

6. Known major head trauma (falls, motor vehicle accident, gunshot wounds, etc)

To facilitate downstream analysis, the anterior and posterior segment are dissected apart during tissue processing.

Collection of post-surgical tissue and fixation.

Collection-Fixation: Fixation of Eye Tissue at UAB

Collection-Freezing: Freezing and Embedding Eye Tissue

Initial Rapid Pathology Assessment of Eye Tissue

Eyes are assessed by ex vivo imaging using optical coherence tomography (OCT), color fundus photography, and near infrared reflectance scanning laser ophthalmoscopy. Normal retinas are those with even and uniform band thicknesses and reflectivity on OCT, with smooth changes in layer thicknesses between retinal regions. In the case of non-uniform band thicknesses and reflectivity levels, eyes that do not exhibit features beyond what can be explained by known post-mortem artifact are considered normal. Known artifacts include detachments of retina from retinal pigment epithelium (RPE), of RPE from choroid, of neurosensory retina from bacillary layer, RPE undulation, cystic change).

Optical Coherence Tomography (OCT)and Scanning Color Fundus Photography: (steps 5-6 of Fixation of Eye Tissue at UAB)

Ocular tissue is further evaluated using histological staining:

PASH Staining

Cryosectioning of tissues into micrometer thick sections, alternating between sections for MALDI, histopathology, MXIF and proteomics.

Sectioning: steps 12-14 of Freezing and Embedding Eye Tissue

LC-based Proteomics

Sample Preparation

Data Acquisition

Data Analysis

Autofluorescence microscopy (performed on all slide-mounted tissue sections)

Pre-IMS Autofluorescence Microscopy

Imaging Mass Spectrometry (IMS)

Matrix Application:

[HTX M5 TM Sprayer](http://dx.doi.org/10.17504/protocols.io.yxmvmnzmbg3p/v3)



[Sublimation](http://dx.doi.org/10.17504/protocols.io.n92ldpyn9l5b/v1)

High resolution IMS data acquisition

Post-IMS Autofluorescence Microscopy and Image Registration

Post IMS AF

Matrix removal and staining

Image registration

Registration of autofluorescence images from both IMS and microscopy sections allows for the direct correlation of two or more orthogonal approaches.

IMS Data Analysis

Data pre-processing

Lipid annotation

LC-MS/MS Lipidomics Analysis

LC-MS/MS from tissue cryosections

Spatial transcriptomics via the Nanostring GeoMx Digital Spatial Profiler

Manual slide preparation

NGS library preparation

Next-generation sequencing