TITLE: VU Biomolecular Multimodal Imaging Center (BIOMIC) Eye characterization pipeline
AUTHORS: Angela R.S.  Kruse, David Anderson, Jeffrey D. Messinger, Jamie Allen, Lukasz Migas, ali.zahraei, ellie.l.pingry, Morad C Malek, Audramjudd, Dongfeng Cao, Melissa Farrow, Christine A. Curcio, Raf Van De Plas, Kevin Schey, Jeff Spraggins

[DESCRIPTION]
We aim to develop high resolution, chemically informative imaging methodologies for building an atlas of human organs, such as the eye.

Scope:
Provide an overview of the methods used by the Vanderbilt Eye Tissue Mapping Center as part of the Human Biomolecular Atlas Program (HuBMAP, NIH Common Fund) and contextualize individual protocols within our larger workflow.


[STEPS]
SECTION: Organ Procurement and Processing
1. 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 
SECTION: Tissue Preparation and Screening
2. 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
SECTION: Tissue Preparation and Screening
3. 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
SECTION: Sample Assays
4. LC-based Proteomics
Sample Preparation
Data Acquisition
Data Analysis
SECTION: Sample Assays
5. Autofluorescence microscopy (performed on all slide-mounted tissue sections)
Pre-IMS Autofluorescence Microscopy
 

SECTION: Sample Assays
6. Imaging Mass Spectrometry (IMS)
Matrix Application:
    HTX M5 TM Sprayer
    Sublimation

High resolution IMS data acquisition
SECTION: Sample Assays
7. 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.
SECTION: Sample Assays
8. IMS Data Analysis
Data pre-processing
Lipid annotation
SECTION: Sample Assays
9. LC-MS/MS Lipidomics Analysis
LC-MS/MS from tissue cryosections
SECTION: Sample Assays
10. Spatial transcriptomics via the Nanostring GeoMx Digital Spatial Profiler
Manual slide preparation
NGS library preparation
Next-generation sequencing