Abstract
Purpose :
High-resolution optical techniques often use high numerical aperture (NA) microscopy with immersion media, such as water or oil, to optimize imaging resolution. However, these techniques present challenges, such as limited working distances and instability of immersion media often caused by drippage or evaporation. These limitations are compounded during long imaging sessions, such as timelapse captures, and during complex imaging procedures unamenable to submersion in liquid, such as imaging in the eye. We address these obstacles and vastly improve the stability and duration of high NA intravital imaging while maintaining excellent resolution by employing cohesive hyaluronan gel (HG) in place of water as immersion media.
Methods :
All imaging was acquired with two-photon excited fluorescence (TPEF) microscopy using a 1.1 NA, 25X water-immersion objective with a correction collar (CC) and a femtosecond laser centered at 970nm. Cranial windows were installed for cerebrovascular imaging. Transscleral imaging was performed in CX3CR-1GFP knock-in mice for timelapse capture of retinal microglia. Imaging performance was compared between water and HG using measures of depth-dependent signal-to-noise ratio (SNR), resolution, and long-term stability.
Results :
For cerebrovascular imaging, the SNR, maximum imaging depth, and resolution of fine capillary features were nearly identical between water and HG. For timelapse imaging of retinal microglia, the SNR and resolution of fine cellular features were also nearly identical between water and HG. However, HG demonstrated superior imaging longevity and stability of up to 45 minutes without reapplication of media, whereas image quality with water deteriorated rapidly after 10 minutes. Imaging resolution between water and HG were comparable by analysis of point-spread-functions (PSF) using 100nm fluorescent beads, and peak resolution of water and HG were equivalent when optimized with the CC.
Conclusions :
We demonstrate the utility of HG as an alternative immersion medium to water that significantly improves the duration and stability of high-resolution imaging. HG is stable during long imaging sessions, such as timelapse of retinal microglia, and demonstrates equivalent imaging quality to water in the context of TPEF microscopy. This approach is practical and drastically improves the feasibility of imaging biological structures with poor access, such as the mouse eye.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.