Hybrid optical gating for long-term 3D time-lapse imaging of the beating embryonic zebrafish heart

Abstract

Three-dimensional fluorescence time-lapse imaging of structural, cellular and subcellular processes in the beating heart is an increasingly achievable goal using the latest imaging and computational techniques. However, previous approaches have had significant limitations. Temporarily arresting the heart using drugs disrupts the heart's physiological state, and the use of ultra-high frame-rates for fluorescence image acquisition causes phototoxic cell damage. Real-time triggered imaging, synchronized to a specific phase in the cardiac-cycle, can computationally "freeze" the heart to acquire the minimal number of fluorescence images required for 3D time-lapse imaging. However, until now no solution has been able to maintain phase-lock to the same point in the cardiac cycle for more than about one hour. Our new hybrid optical gating system maintains phase-lock for up to 24 h, acquiring synchronized 3D+time video stacks of the unperturbed heart in vivo. This approach has enabled us to observe detailed developmental, structural, cellular and subcellular processes, including live cell division and cell fate tracking, in the embryonic zebrafish heart using transgenic fish lines expressing cell-specific fluorophores. We show that our approach not only provides high spatial and temporal resolution 3D-imaging, but also avoids phototoxic injury, where alternative approaches induce measurable harm. This provides superb cellular and subcellular imaging of the heart while it is beating in its normal physiological state, and opens up new and exciting opportunities for further study in the heart and other moving cellular and subcellular structures in vivo.

Tissue clearing and imaging methods for cardiovascular development

“…The ideal model for using live imaging protocols is zebrafish, commonly used for studying cardiac morphogenesis as well as hemodynamics (Boselli and Vermot, 2016;Forouhar et al, 2006;Mellman et al, 2012;Taylor et al, 2019;Truong et al, 2011). The zebrafish eggs are externally fertilized; embryos are nearly transparent, providing optical access to the earliest stages of cardiogenesis (Stainier et al, 1996); and many GFP-labeled transgenic strains have been derived (D'Amico et al, 2007;Lawson and Weinstein, 2002;Nguyen et al, 2008).…”

Deep learning-based framework for cardiac function assessment in embryonic zebrafish from heart beating videos

“…Among animal models used in cardiovascular research, zebrafish (Dario rerio) has been proven to be the premier model for studies of developmental genetics and functional genomics owing to their conserved genome, small size, low-cost for maintenance, short generation time, and optical transparency, just to name a few [2]. In addition, zebrafish cardiac physiology shows similar phenotypes to that of humans [3] and the time-lapse videos of the heart development can be easily acquired [4]. All these make zebrafish an ideal choice to investigate cardiac development, congenital heart disease as well as therapeutic potentials.…”

Highly swelling pH-responsive microgels for dual mode near infra-red fluorescence reporting and imaging

“…Fluorescent reporting and imaging are invaluable tools for remotely studying complex environments such as those within gels and the body. [1][2][3][4][5][6][7] Fluorescence-based technologies provide relatively simple and inexpensive operating procedures whilst delivering real-time imaging and diagnostic data 8,9 with high sensitivity, 10,11 low background noise 12 and potentially low-cost imaging reagents. 13,14 The reporter probes studied to date include organic dyes, 15 uorescent dots, 16 plasmonic nanomaterials, 17 uorescent proteins 18 and upconverting nanoparticles.…”