Advanced Light and Electron Microscopy (ZBS 4), Robert Koch Institute, Berlin, Germany.
J Microsc. 2019 May;274(2):92-101. doi: 10.1111/jmi.12788. Epub 2019 Mar 12.
High-pressure freezing limits the size of biological samples, because only small samples can be frozen without ice damage. Additionally, these samples must fit into the dimensions of the sample holder provided by the high-pressure freezer. We explored the potential of a 10 μm thin polyester filter membrane (PE-filter) as a versatile sample substrate for high-pressure freezing. Planktonic bacteria, bacterial spores and suspended eukaryotic cells could be concentrated on the PE-filter, whereas biofilm, bacterial microcolonies and HeLa cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters were used for high-pressure freezing, freeze-substitution and plastic embedding in Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate as well as the overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms. LAY DESCRIPTION: In order to investigate biological samples in the transmission electron microscope it is imperative to remove all their water content, or the specimens will be destroyed by boiling in the high vacuum of the microscope. In order to avoid dramatic morphology-changes due to drying artefacts or the impact of chemical stabilisers, high-pressure freezing (HPF) was developed. This protocol allows freezing biological samples in an instant (within a few milliseconds) down to -196°C while applying high pressure at the same time so that the specimen retains all its water in a solidified noncrystalline form. However, the formation of morphology-destroying ice crystals is only avoided, if the cooling of the sample is faster than the ice crystal formation, which is only possible with very thin samples (up to a maximum of 200 μm in optimal cases). High-pressure freezing is regarded as the gold-standard for sample preparation of cells, tissues and small organisms. However, all of these samples must fit into the dimensions of the specific sample holder of the high-pressure freezer and their transfer into the high-pressure freezing machine must be achieved without significant impact on sample physiology. Additionally, it may also necessary to concentrate and immobilise a biological specimen before they can be placed in the HPF sample holder. Although a few number of strategies and sample substrates have been used for different types of biological samples, we explored the potential of a 10 μm thin polyester filter membrane (PE-filter) as a versatile sample substrate for HPF. In culture medium suspended bacteria, suspended bacterial spores and in medium suspended higher cells could be concentrated on the PE-filter, whereas bacterial biofilm or bacterial microcolonies from an agar plate, and surface-adhering higher cells were able to grow directly on the PE-filter. These microorganism-loaded PE-filters could be directly used for high-pressure freezing, and were finally embedded in a plastic resin like Epon or Lowicryl. Embedded filters were cross-sectioned so that the interface between microorganism and substrate or overlying medium was revealed. Although the structural preservation was good for thin samples and samples with lower water content, such as biofilms, adherent HeLa-cell cultures were likewise sufficiently preserved for transmission electron microscopy imaging. The fact that microorganism-loaded PE-filters could be also examined with confocal laser scanning fluorescence microscopy under fully hydrated conditions, and freeze-substituted PE-filters samples with scanning-electron microscopy, demonstrates the versatility of the PE-filter as a sample substrate for a wide array of microorganisms.
高压冷冻限制了生物样本的大小，因为只有小样本才能在不损坏冰的情况下进行冷冻。此外，这些样本必须适应高压冷冻机提供的样本架的尺寸。我们探索了 10μm 厚的聚酯过滤膜（PE 滤膜）作为高压冷冻多功能样本基底的潜力。浮游细菌、细菌孢子和悬浮真核细胞可以浓缩在 PE 滤膜上，而生物膜、细菌微菌落和 HeLa 细胞可以直接在 PE 滤膜上生长。这些载有微生物的 PE 滤膜可用于高压冷冻、冷冻置换和包埋在环氧树脂或 Lowicryl 中。嵌入的过滤器被横切，以便显示微生物和基底之间的界面以及覆盖的介质。尽管对于薄样本和含水量较低的样本（如生物膜），结构保存良好，但附着的 HeLa 细胞培养物同样可以进行透射电子显微镜成像。事实上，载有微生物的 PE 滤膜可以在完全水合的条件下用共聚焦激光扫描荧光显微镜进行检查，用扫描电子显微镜检查冷冻置换的 PE 滤膜样品，这证明了 PE 滤膜作为广泛微生物的样本基底的多功能性。
为了在透射电子显微镜下研究生物样本，必须去除所有的水分，否则样本将在显微镜的高真空下被煮沸破坏。为了避免因干燥伪影或化学稳定剂的影响而导致形态发生剧烈变化，开发了高压冷冻（HPF）。该方案允许将生物样本在几毫秒内快速冷冻至-196°C，同时施加高压，从而使样本保留其所有水分，呈固态非晶形。然而，只有在样品的冷却速度快于冰晶形成速度的情况下，才能避免形态破坏冰晶的形成，这种情况只有在非常薄的样品（在最佳情况下最多 200μm）中才有可能。高压冷冻被认为是细胞、组织和小生物样本制备的金标准。然而，所有这些样本都必须适应高压冷冻机特定样本架的尺寸，并且在不显著影响样本生理学的情况下，将其转移到高压冷冻机中。此外，在将生物样本放入 HPF 样本架之前，可能还需要对其进行浓缩和固定。虽然已经有几种策略和样本基底用于不同类型的生物样本，但我们探索了 10μm 厚的聚酯过滤膜（PE 滤膜）作为 HPF 多功能样本基底的潜力。在悬浮培养基中的悬浮细菌、悬浮细菌孢子和悬浮高等细胞可以浓缩在 PE 滤膜上，而来自琼脂平板的细菌生物膜或细菌微菌落以及表面附着的高等细胞可以直接在 PE 滤膜上生长。这些载有微生物的 PE 滤膜可以直接用于高压冷冻，最后嵌入环氧树脂或 Lowicryl 等塑料树脂中。嵌入的过滤器被横切，以便显示微生物和基底或覆盖的介质之间的界面。尽管对于薄样本和含水量较低的样本（如生物膜），结构保存良好，但附着的 HeLa 细胞培养物同样可以进行透射电子显微镜成像。事实上，载有微生物的 PE 滤膜可以在完全水合的条件下用共聚焦激光扫描荧光显微镜进行检查，用扫描电子显微镜检查冷冻置换的 PE 滤膜样品，这证明了 PE 滤膜作为广泛微生物的样本基底的多功能性。
