Apr 11, 2023
VU Biomolecular Multimodal Imaging Center (BIOMIC) Eye characterization pipeline
- Angela R.S. Kruse1,
- David Anderson1,
- Jeffrey D. Messinger2,
- Jamie Allen1,
- Lukasz Migas3,
- Ali Zahraei1,
- Ellie Pingry1,
- Morad C Malek1,
- Audramjudd1,
- Dongfeng Cao4,
- Melissa Farrow1,
- Christine A. Curcio2,
- Raf Van De Plas3,
- Kevin Schey1,
- Jeff Spraggins1
- 1Vanderbilt University;
- 2University of Alabama at Birmingham;
- 3Delft University of Technology;
- 4University of Alabama, Birmingham
Protocol Citation: Angela R.S. Kruse, David Anderson, Jeffrey D. Messinger, Jamie Allen, Lukasz Migas, Ali Zahraei, Ellie Pingry, Morad C Malek, Audramjudd, Dongfeng Cao, Melissa Farrow, Christine A. Curcio, Raf Van De Plas, Kevin Schey, Jeff Spraggins 2023. VU Biomolecular Multimodal Imaging Center (BIOMIC) Eye characterization pipeline. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk49w5g5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 12, 2021
Last Modified: October 18, 2023
Protocol Integer ID: 46201
Keywords: HuBMAP, BIOMIC, Vanderbilt, Multimodal Imaging, IMS, MxIF, Proteomics, Image Registration, Eye
Abstract
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.
Organ Procurement and Processing
Organ Procurement and Processing
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.
Figure 1. Division of the anterior and posterior segment during tissue processing. The anterior segment contains the cornea, iris, ciliary body, and lens. The posterior segment contains the retina, fovea, choroid, sclera, and optic nerve head.
Collection of post-surgical tissue and fixation.
Collection-Fixation: Fixation of Eye Tissue at UAB
Tissue Preparation and Screening
Tissue Preparation and Screening
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:
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
Sample Assays
Sample Assays
Autofluorescence microscopy (performed on all slide-mounted tissue sections)
CITATION
Imaging Mass Spectrometry (IMS)
Matrix Application:
Post-IMS Autofluorescence Microscopy and Image Registration
CITATION
Registration of autofluorescence images from both IMS and microscopy sections allows for the direct correlation of two or more orthogonal approaches.
LC-MS/MS Lipidomics Analysis
Spatial transcriptomics via the Nanostring GeoMx Digital Spatial Profiler