Unequivocal Cardiac Phase Sorting From Alternating Ramp-And Pulse-Illuminated Microscopy Image Sequences

In vivo microscopy is an important tool to study developing organs such as the heart of the zebrafish embryo but is often limited by slow image frame acquisition speed. While collections of still images of the beating heart at arbitrary phases can be sorted to obtain a virtual heartbeat, the presence of identical heart configurations at two or more heartbeat phases can derail this approach. Here, we propose a dual illumination method to encode movement in alternate frames to disambiguate heartbeat phases in the still frames. We propose to alternately acquire images with a ramp and pulse illumination then sort all successive image pairs based on the ramp-illuminated data but use the pulse-illuminated images for display and analysis. We characterized our method on synthetic data, and show its applicability on experimental data and found that an exposure time of about 7% of the heartbeat or more is necessary to encode the movement reliably in a single heartbeat with a single redundant node. Our method opens the possibility to use sorting algorithms without prior information on the phase, even when the movement presents redundant frames.

1. Introduction

Live imaging of organ development is an essential tool to study the beating heart in zebrafish. However, imaging is challenging because the heart is beating starting from early stages of development and requires a fast acquisition frame rate. Fluorescence microscopy is particularly affected as fairly long exposure times are necessary or camera speed and data-transfer bandwidth are limited.