Noninvasive, Label-Free Imaging of Freely Moving <i>C. elegans</i> Nematodes

Noninvasive, label-free imaging of freely moving nematodes offers a powerful but technically demanding approach for behavioral and morphological studies. Key challenges arise from their microscopic size, rapid and intricate locomotion patterns, uniform soft-tissue composition with low intrinsic contrast, and the stringent requirements for imaging systems capable of simultaneously achieving high spatiotemporal resolution and exceptional soft-tissue sensitivity. To overcome these challenges, we employed ultrafast phase-contrast x-ray imaging (PCXI) and phase-contrast computed tomography (PCCT). PCXI was conducted at a detector acquisition speed of 500 fps, while PCCT leveraged a synchronized system comprising a fast-acquisition detector (4000 fps), a rapid-rotation stage ( <a:math xmlns:a="http://www.w3.org/1998/Math/MathML"> <a:mrow> <a:msup> <a:mn>1800</a:mn> <a:mo>∘</a:mo> </a:msup> <a:mtext>/</a:mtext> <a:mi mathvariant="normal">s</a:mi> </a:mrow> </a:math> ), and a precision electromechanical shutter (3-ms actuation). This configuration enabled complete three-dimensional (3D) tomographic acquisition (400 projections over <c:math xmlns:c="http://www.w3.org/1998/Math/MathML"> <c:msup> <c:mn>180</c:mn> <c:mo>∘</c:mo> </c:msup> </c:math> ) in just 100 ms. Our integrated imaging platform delivers dual capabilities: PCXI enables real-time monitoring of multiple nematodes' natural behaviors, including dynamic irradiation avoidance responses, while PCCT offers comprehensive whole-organism morphometric analysis—quantifying body length, thickness, and volume—all under physiologically natural conditions without the need for anesthesia or contrast agent staining. By synergistically integrating ultrafast PCXI for dynamic two-dimensional functional analysis with PCCT for comprehensive 3D morphological characterization, this multimodal platform establishes an imaging paradigm that significantly advances the study of fast-moving microscopic organisms.