Haeri M et al. (2018), Retinal tissue preparation for high-resolution ...

XB-ART-55367

MethodsX 2018 Mar 16;5:1140-1147. doi: 10.1016/j.mex.2018.03.001.

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Retinal tissue preparation for high-resolution live imaging of photoreceptors expressing multiple transgenes.

Haeri M , Zhuo X , Haeri M , Knox BE .

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Live imaging has become the favorite method in recent years to study the protein transport, localization and dynamics in live cells. Protein transport is extremely essential for proper function of photoreceptors. Aberration in the proper transport of proteins gives rise to the loss of photoreceptor and blindness. On the other hand, the ease of generation of transgenic Xenopus laevis tadpoles and the advantage of high resolution live confocal imaging provide new insight into understanding protein dynamics in photoreceptors. There are several steps for quantifying and visualizing fluorescently tagged proteins in photoreceptors starting with assembly of plasmids, generation of transgenic tadpoles, preparation of retinal tissues, imaging the transgenic photoreceptors and finally analyzing the recorded data. The focus of this manuscript is to describe how to prepare retinal tissues suited for live cell imaging and provide our readers with a tutorial video. We also give a summary of steps leading to a successful experiment that might be designed for imaging the ultrastructures of photoreceptors, the expression of two or more different fluorescently tagged proteins, their localization, distribution, or protein dynamics within photoreceptors. •Retinal tissue live imaging demonstrates the ultrastructures of photoreceptors.•High resolution live confocal imaging provides new insight into understanding the pathophysiology of photoreceptors.

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Species referenced: Xenopus laevis
Genes referenced: arrb2 rho

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	Fig. 1. (A) A healthy live Xenopus laevis retinal tissues with healthy cells imaged with confocal microscope while bathed with frog ringer solution. (B) Live retinal tissue prepared from transgenic frogs expressing soluble eGFP. Using AxioVision 4.8 a surface was rendered on florescent cells and by rotating the x, y and z planes part of the surface was removed. The arrow shows the axoneme. (C) Live piece of retinal tissue prepared from transgenic frogs expressing an eGFP tagged mutant opsin (P23H [11]). The distribution of tagged mutant rhodopsin in both inner and outer segment can be seen clearly. Some fluorescent foci resembling aggregates are seen in the outer segment. (D) A single live photoreceptor expressing eGFP tagged arrestin and mCherry tagged rhodopsin (Rho-mCherry), simultaneously. The Rho-mCherry is localized to the outer segment and the eGFP tagged arrestin is localized to the inner segment mostly while it can also be detected in the axoneme (arrow). (E) A high-frequency fluorescent banding caused by light-dark cycle is seen in a rod photoreceptor expressing rhodopsin-eGFP. Dark lines along the rod long axis are generated by incisures which are invaginations of the disk membrane deprived of rhodopsin [23]. (F) The expression of an eGFP-tagged mutant rhodopsin (P23H) results in the aberrant expression of mutant protein in the inner segment and the formation of aggregates (*). OS: Outer segment IS: inner segment. Scale bar is 5 μm.
	Fig. 2. (A–C) A single rod photoreceptor expressing a tagged eGFP that is targeted to the mitochondrial membrane. Higher resolution of the photoreceptor ellipsoid followed by deconvolution demonstrates the distribution of the tagged eGFP in the mitochondrial membrane (arrow). (D & E) The depression in the outer membrane layer covering the outer segment is caused by incisures. This depression can be seen as a dark line along the rod axis with live imaging of freshly isolated photoreceptors (arrow). (F & G) The base of a rod photoreceptor expressing rhodopsin-eGFP. A pack of disks attached to the outer segment and inner segment boundary is seen that contains eGFP (*). (H) Photobleaching of a rod photoreceptor expressing double geranylated eGFP shows the outer segment before photobleaching (H1), right after photobleaching (H2) and 50 s after photobleaching (H3). The double geranylated eGFP is free to diffuse laterally (within the disks and along the rod short axis) and therefore it demonstrates the recovery after photobleaching along the disk axis. There is minimal recovery along the rod long axis, on the other hand, due to trivial movement of double geranylated eGFP along the rod axis.

References [+] :

Aerni-Flessner, Can fly photoreceptors lead to treatments for rho ((P23H)) -linked retinitis pigmentosa? 2013, Pubmed