Light‐sheet Imaging to Characterize Vascular Development in Murine Retina

Abstract

The advent of light-sheet fluorescence microscopy (LSFM) has revolutionized the imaging of the cardiovascular system, allowing for visualizing the 3-D intact vascular network with high-spatiotemporal resolution and minimal photobleaching. We introduce the custom-built LSFM combined with simplified passive CLARITY (SPC) method to study the vascular development in murine retina bypassing the flat-mount sample preparation. This imaging strategy enables the rapid acquisition of entire retina with a single scan to interrogate vascular development and study angiogenesis. For the sample preparation, we dissected the retina from C57BL/6 mice at P10 for clearing. The clearing method was modified based on the previous passive CLARITY method. In brief, mouse retinas were fixed in 4% Paraformaldehyde (wt/vol) for 2 hours and transferred to the Eppendorf filled with monomer solution (4% Acrylamide (wt/vol), 0.05% Bis-Acrylamide (wt/vol), and 0.25% VA-044 initiator (wt/vol) in PBS) and incubated overnight. After thorough incubation, we put the sample in 37 degrees water bath for hydrogel polymerization for 4 hours. The retinal were then placed into a clearing solution comprised of 4% w/v sodium dodecyl sulfate (SDS) and 1.25% w/v boric acid (pH 8.5). The samples were incubated at 37 °C until cleared (Figure 1B). Our clearing method allowed for the generation of tissue-hydrogel hybrids to support the intact bowl shape structure of the retina. After the SPC, the retinas were washed with PBS and incubated with Biotinylated GS I4B lectin in 1:20 dilution for overnight and secondary antibody Alexa-488 conjugated streptavidin with 1:100 dilution for 2 days to complete the staining on vasculature. Our in-house dual-sided illumination LSFM applied a continuous wave laser with triple wavelengths as the illumination source. The detection module was installed perpendicular to the illumination plane, and it was composed of a stereo microscope with a 1X magnification objective (NA = 0.25), a scientific CMOS and a set of filters (Figure 1A). The lateral resolution and axial resolution of this LSFM system are 3.25 μm and 5 μm respectively. The cleared retinas were immersed in the refractive index (RI) matching solution (RI: 1.46–1.4844) with 1% agarose solution was mounted in a Borosilicate glass tubing (RI = 1.47) to reduce refraction and reflection among various interfaces. We succeeded in imaging the 3-D vascular network (Figure 1C), demonstrating the feasibility of this strategy to provide a structural analysis of the microvascular development. In addition, the adapted SPC preserved the native structure of the retina comparing to the flat-mount. It further reduces the complexity for clearing by removing vacuum chamber and nitrogen tank. This advanced imaging strategy is not limited to the shallow primary plexus layer for morphometric measurements but providing an accessibility for investigating the deeper secondary vascular plexus in murine. Unlike traditional confocal or wide-field imaging of the flat-mount retina, LSFM allows for rapid scanning with high axial resolution and low photo-bleaching, enabling 3D spatial localization of the tissue structure and cellular events with multi-channels of fluorescence. Support or Funding Information NIH (5R01HL083015-10) Figure 1Open in figure viewerPowerPoint A. Schematic diagram of a dual-sided illumination for the light-sheet fluorescent microscope. B. P10 mouse retinas before SPC (left), after clearing was complete (right) at day 7 of the clearing. C. 3-D perception of Alexa-488 labeled retina samples in ROI displaying the 3D vascular network. Scale bar: (B) 1 mm (C) 200 μm for. M1-10: mirror; PH: pinhole; ND: neutral density filter; BE: beam expander; S: slit; CL1-2: cylindrical lens; L1-4: achromatic doublets; Ob1-2: objective This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.