Wang Yao L, Grooms Noa W F, Jaklitsch Erik L, Schulting Leilani G, Chung Samuel H
Department of Bioengineering, Northeastern University, Boston, MA 02115, USA.
iScience. 2023 Jan 18;26(2):105999. doi: 10.1016/j.isci.2023.105999. eCollection 2023 Feb 17.
Despite its profound impact on biology, high-resolution microscopy largely remains low throughput because current immobilization techniques require substantial manual effort. We implement a simple cooling approach to immobilize entire populations of the nematode directly on their cultivation plates. Counterintuitively, warmer temperatures immobilize animals much more effectively than the colder temperatures of prior studies and enable clear submicron-resolution fluorescence imaging, which is challenging under most immobilization techniques. We demonstrate 64× z-stack and time-lapse imaging of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. High-throughput imaging of a fluorescent proxy in cooled animals and direct laser axotomy indicate that the transcription factor CREB underlies lesion conditioning. By obviating individual animal manipulation, our approach could empower automated imaging of large populations within standard experimental setups and workflows.
尽管高分辨率显微镜对生物学有着深远影响，但由于当前的固定技术需要大量人工操作，其通量在很大程度上仍然较低。我们实施了一种简单的冷却方法，可将线虫的整个群体直接固定在其培养板上。与直觉相反，较温暖的温度比先前研究中的较低温度能更有效地固定动物，并能实现清晰的亚微米分辨率荧光成像，而这在大多数固定技术下都具有挑战性。我们展示了对成虫和胚胎中的神经元进行64倍z轴堆叠和延时成像且无运动模糊。与标准叠氮化物固定相比，冷却固定将动物制备和恢复时间减少了98%以上，显著提高了实验速度。对冷却动物中荧光替代物的高通量成像以及直接激光轴突切断表明，转录因子CREB是损伤条件作用的基础。通过避免对单个动物的操作，我们的方法能够在标准实验设置和工作流程中实现对大量群体的自动成像。
