Jan De Bock , Dr.

Application Manager (Life Science Research), Leica Microsystems CMS GmbH.

Jan has worked as a microscope professional in different roles since 2003. He joined Leica Microsystems in 2011 as a product specialist for confocal microscopy. In 2017, he became a member of the application management team where he is also responsible for correlative workflows, involving microscope systems under cryogenic conditions and sample preparation equipment. Jan studied biology and earned his PhD in the field of olfactory research.

Mouse hippocampus brain slice on a grid after HPF using the “Waffle Method”.

The “Waffle Method”: High-Pressure Freeze Complex Samples

This article describes the advantages of a special high pressure freezing method, the so-called “Waffle Method”. Learn how the “Waffle Method” uses EM grids as spacers for high-pressure freezing,…

C. elegans embedded in Lowicryl® HM20; pharynx showing red fluorescence (mCherry). The overview shows a front view onto the resin capsule formed by the bottom of a flow-through chamber of the EM AFS2. The capsule was pretrimmed manually. The blockface was trimmed automatically using the AutoTrim function of UC Enuity guided by fluorescence of the worm. Edge length of both squares in relation to the images is 250 µm.

How Fluorescence Guides Sectioning of Resin-embedded EM Samples

Electron microscopes, including transmission electron microscopes (TEM) and scanning electron microscopes (SEM), are widely utilized to gain detailed structural information about biological samples or…

如何通过自动化超薄切片技术节省时间与样本

本文阐述了如何利用树脂包埋电镜样本的 3D micro-CT 数据，在切片前将样本修整至预设目标平面。采用Leica UC Enuity 系统的交互式自动化方案，可显著节省时间、减少样本损耗及缩短新手用户的培训周期。

Block-face created by automatic trimming under fluorescence. Mammalian cells of interest, stained with CellTrackerTM Green are visualized within the block-face using the UC Enuity equipped with the stereo microscope M205 FA. In the background a carbon finder grid in black is visible. All samples in the article are created by Felix Gaedke, PhD, CECAD, Cologne, Germany.

如何在块面中自动获取感兴趣的荧光细胞

本文介绍了使用超薄切片超薄切片机自动修整修块功能，获取树脂块面中带有荧光信号的细胞结构。我们展示了如何使用配置有体视显微镜 M205 FA 的超薄切片超薄切片机 UC Enuity ，来识别感兴趣的荧光细胞，如何自动修整包含细胞的块面，以及如何在切片中观察细胞而无需转移到外部显微镜。

Camera image during auto alignment. The feedback lines indicate if the correct edges in the image are detected. Green: Vertical center line; Magenta: Upper edge of the light gap; White: Lower edge of the light gap (not visible here, falling together with red line); Red: Knife edge; Blue: Left and right edge of the block face being automatically detected.

高质量超薄切片：样品与切片刀自动对齐

超薄切片技术是获取样品切片的最常用方法。在室温条件制备时，将样品小块嵌入环氧树脂中，然后通过修剪去除多余的树脂，并使用玻璃刀或金刚石刀将样品切成厚度为50-100纳米之间的薄片。

Projection of a confocal z-stack. Sum159 cells, human breast cancer cells kindly provided by Ievgeniia Zagoriy, Mahamid Group, EMBL Heidelberg, Germany. Blue–Hoechst - indicates nuclei, Green–MitoTracker mitochondria, and red–Bodipy - lipid droplets

低温光学显微镜的新成像工具

荧光显微镜图像能够为cryo-FIB加工提供定位支持，其质量决定了所制备薄片的结果。本文描述了LIGHTNING技术是如何显著提高图像质量，以及如何利用该技术基于荧光寿命的信息来辨别样品的不同结构。

Correlation of markers in the LM and the FIB image.

如何对荧光结构三维定位以进行冷冻FIB切片

冷冻ET（电子断层扫描）是一种专用的透射电子显微镜技术，可以重建观察区域的三维体积。借助先进的冷冻EM（电子显微镜），图像分辨率可以提升到令人难以置信的亚纳米等级。因此，可以在细胞内的原生环境中研究蛋白质以及其他生物分子，从而揭示尚未探明的分子机制。由于细胞和组织必须薄到能够透过电子，样品必须进行切片以获取足够薄的样品体积（薄层）。为对样品中的靶区进行精确的三维定位，冷冻共聚焦显微镜是必不可少的工…

HeLa Kyoto cells (HKF1, H2B-mCherry, alpha Tubulin, mEGFP). Left image: Maximum projection of a z-stack prior to ICC and LVCC. Right image: Maximum projection of a mosaic z-stack after ICC and LVCC.

如何使用Coral Life（活细胞光电联用）改进活细胞成像

对于活细胞 CLEM 应用而言，光学显微镜成像是在正确的时间以正确的状态识别正确细胞的关键步骤。在本文中，徕卡专家就使用宽场系统的优势以及使用蓝宝石作为细胞培养基底时需要克服的障碍分享了他们的见解。

Cryo FIB lamella - Overlay of SEM and confocal fluorescence image. Target structure in yeast cells (nuclear pore proteine Nup159-Atg8-split Venus, red) marked by an arrow. Scale bar: 5 µm. Alegretti et al., Nature 586, 796-800 (2020).

使用冷冻共聚焦显微镜定位活性循环核孔复合物

本文介绍了如何利用冷冻光学显微镜，尤其是冷冻共焦显微镜来提高冷冻工作流程的可靠性。评估了EM网格和样品的质量，并分析了目标结构的分布。本文展示了如何将冷冻共焦3D数据投射到SEM图像上，将感兴趣结构可靠地保留在FIB切割的薄片内，以便在冷冻TEM中进行进一步研究。

Scheme of a 2D mosaic scan. Drosophila melanogaster (eye section)

马赛克图片

共焦激光扫描显微镜被广泛用于生成细胞、亚细胞结构甚至单分子的高分辨率三维图像。不过，越来越多的科学家正将生物研究的重点从单细胞研究扩展到整个器官和生物体，分析动物体内复杂的相互作用。