Microsurgical Dissection and Tissue Clearing for High Resolution Intact Whole Retina and Vitreous Imaging

Summary

Presented here is a protocol for intact whole retina imaging in which the outer opaque/pigmented layers of the eyeball are surgically removed, and optical clearing is applied to render retina transparent enabling the visualization of the peripheral retina and hyaloid vasculature in intact retina using light sheet fluorescent microscopy.

Abstract

Neuronal and vascular structures of the retina in physiologic and pathologic conditions can be better visualized and characterized by using intact whole retina imaging techniques compared to conventional retinal flat mount preparations and sections. However, immunofluorescent imaging of intact whole retina is hindered by the opaque coatings of the eyeball, i.e., sclera, choroid, and retinal pigment epithelium (RPE) and the light scattering properties of retinal layers that prevent full thickness high resolution optical imaging. Chemical bleaching of the pigmented layers and tissue clearing protocols have been described to address these obstacles; however, currently described methods are not suitable for imaging endogenous fluorescent molecules such as green fluorescent protein (GFP) in intact whole retina. Other approaches bypassed this limitation by surgical removal of pigmented layers and the anterior segment of the eyeball allowing intact eye imaging, though the peripheral retina and hyaloid structures were disrupted. Presented here is an intact whole retina and vitreous immunofluorescent imaging protocol that combines surgical dissection of the sclera/choroid/retina pigment epithelium (RPE) layers with a modified tissue clearing method and light sheet fluorescent microscopy (LSFM). The new approach offers an unprecedented view of unperturbed vascular and neuronal elements of the retina as well as the vitreous and hyaloid vascular system in pathologic conditions.

Introduction

The interaction between the retinal neuronal and vascular elements in healthy and disease states is traditionally explored by immunofluorescent studies on physical sections of paraffin- or cryo-fixed retina tissue or on retina flat preparations¹. However, tissue sectioning disrupts retina neuronal and vascular continuity, and although three-dimensional reconstruction of the adjacent retina sections is suggested as a possible solution, it is still subject to errors and artifacts. Retina flat mount preparations also markedly disturb the integrity of retinal vascular and neuronal elements and the geographic connection between adjacent retinal area....

Protocol

All experiments were approved by the University of Texas Medical Branch Institutional Animal Care and Use Committee (IACUC). Animal use and care were in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statement for use of animals in ophthalmic and vision research. All the materials required to carry out this procedure are listed in the Table of Materials. Wear powder-free gloves while performing each step. For steps 6 and 7, also refer to the official microscope operating .......

Discussion

Retina and vitreous development and pathologies are best studied with intact whole retina imaging techniques in which the retina is not cut for sections or for flat mount preparations. Existing intact whole eye imaging methods either incorporate pigment bleaching, which removes innate fluorophores, or involve physical removal of the opaque coatings of the eyeball (RPE, choroid, and sclera) along with the anterior segment of the eye, which may disturb peripheral retina and vitreous body. Chang et al. and Prahst et al. rem.......

Materials

Name	Company	Catalog Number	Comments
Experimental animal
CX3CR1-GFP Mouse	The Jackson Laboratory	5582
Anesthetic
Dexmedetomidine	Par Pharmaceutical	42023-146-25
Ketamine	Fresenius Kabi
Tissue harvesting, fixation, and sample dissection
cardiac perfusion pump	Fisher scientific	NC9069235
Cyanoacrylate superglue	amazon.com
Fine scissors-sharp	Fine Science Tools	14160-10
Fine tweezers	Fine Science Tools	11412-11
Paraformaldehyde (PFA)	Electrone microscopy sciences	15710-S
Phosphate buffered saline (PBS)	Gibco	10010049
size 1 painting brush	dickblick.com
straight spring scissors	Fine Science Tools	15000-03
syringe, needle tip, 27 gauge x 1.25"	BD
Tubes 1.5 ml, 15 ml, 50 ml	Thermo sceintific
Tween-20	ThermoFisher	85114
Immunofluorescent staining
Anti-mouse collagen IV antibody	Abcam	ab19808	1:200 dilution
Anti-rabbit Alexa Fluor 568	Invitreogen	A-11011	1:200 dilution
Normal goat serum	ThermoFisher	50062Z	10% concentration
Tissue clearing
2,2′-thiodiethanol (TDE)	Fluka analytica	STBD7772V
Rocking shaker	Fisher scientific	02-217-765
Microscopy
Fluorescent microspheres	TetraSpeck	T14792
Light sheet fluorescent microscope (LSFM)	Zeiss	Z1
Microglia enumeration
ImageJ	National Institue of Health