显微镜知识库

徕卡显微系统的知识库提供有关显微镜学科的科学研究和教学材料。内容旨在对显微镜初学者、有经验的显微镜操作实践者和使用显微镜的科学家在他们的日常工作和实验有所帮助。这里有探索交互式教程和应用笔记，你可以找到你需要的显微镜的基础知识以及前沿技术——快来加入徕卡显微知识社区，分享您的专业知识！

Serial Lift-Out workflow for cryo-ET lamella preparation - Schematic illustration of the Serial Lift-Out method applied to a vitrified biological sample (e.g., C. elegans larva) embedded in bulk vitreous ice (blue). Image courtesy: Prof. Dr. Jürgen M. Plitzko - doi.org/10.1038/s41592-023-02113-5.

Cryo-ET Sample Preparation: From Waffle Method to Serial Lift-Out

Cryo-ET sample preparation becomes more demanding when specimens are thicker, larger, or more complex. This webinar brings together four perspectives on how high-pressure freezing can be connected…

Waffle method lamella preparation and cryo-ET results - Representative workflow and results of lamella preparation from a vitrified C. elegans L1 larva using the waffle method. Image courtesy: Prof. Dr. Jürgen M. Plitzko - doi.org/10.1038/s41592-023-02113-5-

Waffle Method Workflow: From HPF to Cryo-ET Lamellae

Waffle freezing provides an HPF-based route to cryo-ET sample preparation. This workflow guide follows the process from grid and carrier assembly to vitrification, cryo-FIB milling, lamella…

3D rendering of a mouse beta cell with mitochondria (blue), insulin SGs (orange), microtubules (red), nucleus (yellow), and plasma membrane (transparent).

High-Pressure Freezing Protocols for Ultrastructural 3D EM

High pressure freezing (HPF) can help preserve hydrated cells and tissues close to their biological state at the moment of immobilization, supporting more reliable ultrastructural interpretation than…

Foraminifera (Ammonia confertitesta) labeled with membrane-permeable calcein, high-pressure frozen in salt water using EM ICE. The sample was cryo-planed and targeted with the M205 on the Cryo-Fluo Enuity, then transferred under cryo conditions to the Cryo-Stellaris for widefield and confocal imaging, revealing details of the staining pattern. Image courtesy: David Evans, University of Southampton.

High-Pressure Freezing for Organoids: Cryo CLEM & FIB Lift Out

Master cryo EM workflow steps for challenging 3D samples: when to choose HPF vs. plunge freezing, reproducible blotting/ice control, contamination aware transfers, Cryo CLEM 3D targeting in organoids,…

Electron microscope (EM) image of a cross section of C. elegans (roundworm). Courtesy of T. Müller-Reichert, MPI-CBG, Dresden, Germany and K. McDonald, University of California, Berkeley, USA.

高压冷冻简介

水是细胞最主要的组成部分，因此对于维持细胞超微结构至关重要。目前，冷冻固定是固定细胞成分，而不导致其显著结构变化的唯一途径。现阶段有两种常见的方法：投入冷冻与高压冷冻固定。

用 GFAP-A647 进行免疫染色并使用THUNDER成像仪组织成像的小鼠脑片。美国费城宾夕法尼亚大学 H. Xu 提供。

神经科学研究指南

神经科学通常需要研究具有挑战性的标本，以更好地了解神经系统和疾病。徕卡显微镜帮助神经科学家深入了解神经元功能。

"Waffle方法"：使用高压冷冻制备复杂样品

本文介绍了一种特殊的高压冷冻方法，即 "Waffle 方法 "的优点。了解 "Waffle 方法 "如何使用电镜载网作为高压冷冻的载体，从而减少样本厚度并支持复杂生物样本的高效低温电子显微镜工作流程。此外，本文还强调了现代 HPF 系统-徕卡微系统公司 EM ICE 的优势，并列举了 EM ICE 用于 "Waffle方法 "的参考文献。

Micrograph of dinoflagellate cells. Scale bar = 1 µm.

高压冷冻技术如何提升海洋微生物分析效果

在本应用示例中，我们展示了高压冷冻、冷冻替代及超薄切片技术在海洋生物学领域的应用，重点针对甲藻的超微结构分析。EM ICE 设备的便携性使得样本采集后可直接在海洋站进行高压冷冻处理。

如何成功进行活细胞光电关联

Coral Life 提供了简化的活细胞 CLEM 解决方案，用于深入了解细胞成分随时间发生的结构变化。除了工作流程手册中描述的技术处理外，本文还提供了成功进行实验的其他知识。