Laser scanning is used in advanced biological microscopy to deliver superior imaging contrast, resolution and sensitivity. However, it is challenging to scale up the scanning speed required for interrogating a large and heterogeneous population of biological specimens or capturing highly dynamic biological processes at high spatiotemporal resolution. Bypassing the speed limitation of traditional mechanical methods, free-space angular-chirp-enhanced delay (FACED) is an all-optical, passive and reconfigurable laser-scanning approach that has been successfully applied in different microscopy modalities at an ultrafast line-scan rate of 1–80 MHz. Optimal FACED imaging performance requires optimized experimental design and implementation to enable specific high-speed applications. In this protocol, we aim to disseminate information allowing FACED to be applied to a broader range of imaging modalities. We provide (i) a comprehensive guide and design specifications for the FACED hardware; (ii) step-by-step optical implementations of the FACED module including the key custom components; and (iii) the overall image acquisition and reconstruction pipeline. We illustrate two practical imaging configurations: multimodal FACED imaging flow cytometry (bright-field, fluorescence and second-harmonic generation) and kHz 2D two-photon fluorescence microscopy. Users with basic experience in optical microscope operation and software engineering should be able to complete the setup of the FACED imaging hardware and software in ~2–3 months.

激光扫描用于先进的生物显微镜，以提供卓越的成像对比度、分辨率和灵敏度。然而，提高对大量且异质的生物样本群或以高时空分辨率捕获高度动态的生物过程所需的扫描速度具有挑战性。绕过传统机械方法的速度限制，自由空间角啁啾增强延迟 (FACED) 是一种全光学、无源和可重构的激光扫描方法，已以超快的线扫描速率成功应用于不同的显微镜模式1–80 MHz。最佳的 FACED 成像性能需要优化的实验设计和实施，以实现特定的高速应用。在这个协议中，我们的目标是传播信息，使 FACED 能够应用于更广泛的成像模式。我们提供 (i) FAED 硬件的综合指南和设计规范；(ii) FACED 模块的分步光学实现，包括关键的定制组件；(iii) 整体图像采集和重建流程。我们说明了两种实用的成像配置：多模态 FACED 成像流式细胞术（明场、荧光和二次谐波产生）和 kHz 2D 双光子荧光显微镜。具有光学显微镜操作和软件工程基本经验的用户应该能够在约 2-3 个月内完成 FACED 成像硬件和软件的设置。我们提供 (i) FAED 硬件的综合指南和设计规范；(ii) FACED 模块的分步光学实现，包括关键的定制组件；(iii) 整体图像采集和重建流程。我们说明了两种实用的成像配置：多模态 FACED 成像流式细胞术（明场、荧光和二次谐波产生）和 kHz 2D 双光子荧光显微镜。具有光学显微镜操作和软件工程基本经验的用户应该能够在约 2-3 个月内完成 FACED 成像硬件和软件的设置。我们提供 (i) FAED 硬件的综合指南和设计规范；(ii) FACED 模块的分步光学实现，包括关键的定制组件；(iii) 整体图像采集和重建流程。我们说明了两种实用的成像配置：多模态 FACED 成像流式细胞术（明场、荧光和二次谐波产生）和 kHz 2D 双光子荧光显微镜。具有光学显微镜操作和软件工程基本经验的用户应该能够在约 2-3 个月内完成 FACED 成像硬件和软件的设置。我们说明了两种实用的成像配置：多模态 FACED 成像流式细胞术（明场、荧光和二次谐波产生）和 kHz 2D 双光子荧光显微镜。具有光学显微镜操作和软件工程基本经验的用户应该能够在约 2-3 个月内完成 FACED 成像硬件和软件的设置。我们说明了两种实用的成像配置：多模态 FACED 成像流式细胞术（明场、荧光和二次谐波产生）和 kHz 2D 双光子荧光显微镜。具有光学显微镜操作和软件工程基本经验的用户应该能够在约 2-3 个月内完成 FACED 成像硬件和软件的设置。