📚 Electron microscopy | 电子显微镜
Electron microscopy has revolutionised our ability to visualise biological structures, extending the range of human observation down to the molecular and even atomic scale. By using a beam of electrons instead of visible light, electron microscopes overcome the fundamental resolution limits imposed by the wavelength of light, revealing intricate details of cells, organelles, and macromolecular complexes that are completely invisible under a light microscope. In the context of A‑Level Biology, understanding how electron microscopy works and how to interpret electron micrographs is essential for connecting structure to function in living systems.
电子显微镜彻底改变了我们观察生物结构的能力，将人类观察的范围扩展到了分子甚至原子尺度。通过使用电子束代替可见光，电子显微镜克服了光波长带来的基本分辨率限制，展现出细胞、细胞器和生物大分子复合物的精细细节，这些在光学显微镜下完全不可见。在A‑Level生物学中，理解电子显微镜的工作原理以及如何解读电子显微图像，对于建立结构与功能之间的联系至关重要。
1. The need for higher resolution | 对更高分辨率的需求
The resolution of a microscope is the smallest distance by which two points can be separated and still be distinguished as separate entities. For light microscopy, the theoretical limit of resolution is about 200 nm, set by the wavelength of visible light (approximately 400–700 nm). Many sub‑cellular structures, such as ribosomes (∼25 nm), membranes (∼7–10 nm) and viruses (∼20–300 nm), fall well below this limit, meaning they cannot be clearly resolved with a light microscope. To visualise such tiny objects, a radiation source with a much shorter wavelength is required.
显微镜的分辨率是指能够将两个点分开并仍能将其区分为独立实体的最短距离。对于光学显微镜，理论分辨率极限约为200纳米，这是由可见光的波长（约400–700纳米）决定的。许多亚细胞结构，如核糖体（约25纳米）、膜（约7–10纳米）和病毒（约20–300纳米），都远低于这个极限，意味着使用光学显微镜无法清晰分辨它们。为了观察这些极小的物体，需要一种波长更短的辐射源。
Electrons, when accelerated by a high voltage, exhibit wave‑like properties with an extremely short wavelength. According to the de Broglie relation, the wavelength λ of an electron is given by λ = h / √(2 m e V), where h is Planck’s constant, m is the electron mass, e is the elementary charge, and V is the accelerating voltage. For an accelerating voltage of 100 kV, the electron wavelength is about 0.004 nm — roughly 100 000 times shorter than that of visible light. This extremely short wavelength is the key to the outstanding resolving power of electron microscopes, which can reach down to 0.1 nm in practice.
电子在高压加速下表现出波动性，且波长极短。根据德布罗意关系，电子的波长λ由λ = h / √(2 m e V)给出，其中h为普朗克常数，m为电子质量，e为基本电荷，V为加速电压。对于100千伏的加速电压，电子波长约为0.004纳米——大约比可见光短10万倍。这种极短的波长是电子显微镜具有卓越分辨率的关键，实际分辨率可低至0.1纳米。
2. Basic principles of electron microscopy | 电子显微镜的基本原理
Instead of glass lenses, an electron microscope uses electromagnetic lenses — coils of wire that generate magnetic fields to focus the electron beam. The entire column must be maintained under a high vacuum to prevent electrons from being scattered by air molecules. Electrons are emitted from a source (an electron gun), accelerated by a high voltage, and then focused by a series of condenser lenses onto the specimen. After interacting with the specimen, the transmitted or emitted electrons are projected by objective and projector lenses to form an image on a fluorescent screen or a digital camera.
电子显微镜使用电磁透镜（即产生磁场来聚焦电子束的线圈）代替玻璃透镜。整个镜筒必须保持在高真空状态，以防止电子被空气分子散射。电子从电子源（电子枪）发射，经高压加速后，由一系列聚光镜聚焦到标本上。与标本相互作用后，透射或发射的电子由物镜和投影镜投射，在荧光屏或数码相机上形成图像。
Resolution in an electron microscope is not limited by electron wavelength alone; it is also affected by lens aberrations (spherical and chromatic) and by the quality of specimen preparation. Modern instruments correct for some of these aberrations and routinely achieve resolutions that make individual heavy atoms visible, a capability that has transformed structural biology.
电子显微镜的分辨率不仅受电子波长限制，还受到透镜像差（球差与色差）以及标本制备质量的影响。现代仪器能够校正部分像差，常规分辨率已可看到单个重原子，这一能力极大地改变了结构生物学。
3. Transmission electron microscope (TEM) | 透射电子显微镜
The transmission electron microscope (TEM) operates by passing a high‑energy electron beam through an ultra‑thin specimen. Electrons that are transmitted through the sample carry information about its internal structure. Dense regions scatter more electrons and appear darker in the final image, while regions that allow electrons to pass through appear brighter. This mode of imaging produces two‑dimensional, high‑resolution projections of thin sections or isolated particles.
透射电子显微镜通过将高能电子束穿过超薄标本来工作。穿过样本的透射电子携带了其内部结构的信息。致密区域会散射更多的电子，在最终图像中显得较暗，而允许电子通过的区域则较亮。这种成像模式可以产生薄切片或分离颗粒的二维高分辨率投影。
TEMs can achieve a point‑to‑point resolution better than 0.2 nm, which is sufficient to resolve the double‑stranded structure of DNA or the lattice arrangement of proteins in a crystal. In biology, TEM is used routinely to study cell ultrastructure — for example, detailing the cristae of mitochondria, the thylakoid stacks in chloroplasts, and the bilayer organisation of membranes.
透射电镜的点对点分辨率优于0.2纳米，足以分辨DNA的双链结构或蛋白质在晶体中的晶格排列。在生物学中，透射电镜常用于研究细胞超微结构——例如，详细显示线粒体的嵴、叶绿体中类囊体堆叠，以及膜的双层结构。
4. Scanning electron microscope (SEM) | 扫描电子显微镜
In scanning electron microscopy, a focused beam of electrons scans the surface of a bulk specimen in a raster pattern. Instead of collecting transmitted electrons, the SEM detects signals generated from the surface, principally secondary electrons and backscattered electrons. Secondary electrons provide topographical contrast, producing images with a strikingly three‑dimensional appearance, while backscattered electrons give compositional information based on atomic number.
在扫描电子显微镜中，聚焦的电子束以光栅模式在块状标本的表面进行扫描。SEM并不收集透射电子，而是检测从表面产生的信号，主要是二次电子和背散射电子。二次电子提供形貌衬度，生成具有明显三维外观的图像，而背散射电子则根据原子序数提供成分信息。
The resolution of an SEM is typically in the range of 1–20 nm, which is lower than that of a TEM but more than adequate for visualising whole cells, tissues, and the surfaces of small organisms. Specimens for SEM do not need to be cut into ultra‑thin sections, which simplifies preparation and allows observation of structures in a more natural 3D context.
SEM的分辨率通常在1–20纳米范围内，低于透射电镜，但对于观察完整细胞、组织和小生物表面来说绰绰有余。SEM的标本无需切成超薄切片，这简化了制备流程，并允许在更自然的三维背景下观察结构。
5. Sample preparation: general challenges | 样本制备：一般性挑战
Biological specimens pose numerous difficulties for electron microscopy: they are mostly composed of light elements (carbon, hydrogen, oxygen, nitrogen) that scatter electrons weakly; they contain a high proportion of water, which would evaporate instantly in the vacuum; and they are susceptible to radiation damage from the energetic electron beam. Therefore, elaborate preparation protocols have been developed to stabilise and contrast biological material while preserving its native structure as much as possible.
生物标本给电子显微镜带来了许多困难：它们主要由散射电子能力弱的轻元素（碳、氢、氧、氮）组成；含有高比例的水分，在真空中会立即蒸发；而且容易受到高能电子束的辐射损伤。因此，人们建立了复杂的制备流程来稳定和衬托生物材料，同时尽量保持其天然结构。
6. TEM sample preparation | 透射电镜样本制备
The classic workflow for TEM includes primary fixation with glutaraldehyde (which cross‑links proteins), secondary fixation with osmium tetroxide (which adds electron‑dense heavy metal atoms to lipids and membranes), dehydration in an ethanol or acetone series, embedding in epoxy resin, and cutting ultra‑thin sections (50–100 nm thick) using an ultramicrotome with a diamond knife. Sections are then collected onto metal grids and stained with heavy‑metal salts such as uranyl acetate and lead citrate to enhance contrast.
经典的透射电镜工作流程包括：用戊二醛进行初步固定（交联蛋白质），用四氧化锇进行后固定（为脂质和膜添加电子致密的重金属原子），在乙醇或丙酮梯度中脱水，用环氧树脂包埋，并使用配备金刚石刀的超薄切片机切出超薄切片（厚50–100纳米）。随后将切片收集到金属载网上，并用醋酸铀酰和柠檬酸铅等重金属盐进行染色，以增强反差。
Rapid freezing techniques, such as plunge‑freezing in liquid ethane, can immobilise biological samples in vitreous ice without the need for chemical fixation or staining. This approach, which underpins cryo‑electron microscopy (cryo‑EM), preserves macromolecular assemblies in a near‑native state and has become transformative for high‑resolution structural biology.
快速冷冻技术，如投入液态乙烷中进行速冻，可以将生物样品固定于玻璃态冰中，无需化学固定或染色。这种方法是冷冻电镜的基础，能够在近乎天然的状态下保存生物大分子复合物，并已在高分辨率结构生物学中引发变革。
7. SEM sample preparation | 扫描电镜样本制备
SEM samples are usually fixed, dehydrated, and then dried by critical‑point drying or freeze‑drying to prevent surface tension forces from collapsing delicate structures. Since biological material is non‑conductive, the dried specimen is typically coated with a thin layer of metal (e.g. gold or gold‑palladium) via sputter coating. This conductive coating prevents the accumulation of negative charge (charging) on the sample surface, which would otherwise distort the image.
SEM样本通常经固定、脱水，然后通过临界点干燥或冷冻干燥进行干燥，以防止表面张力使脆弱的结构塌陷。由于生物材料不导电，干燥后的标本通常通过溅射镀膜法镀上一层薄金属层（如金或金‑钯）。这种导电涂层能防止样本表面负电荷的积累（充电现象），否则会扭曲图像。
8. Comparing TEM and SEM | 透射电镜与扫描电镜的比较
The table below summarises the key differences between the two main types of electron microscopes used in biology.
下表总结了生物学中使用的两种主要电子显微镜之间的主要区别。
| Feature | TEM | SEM |
|---|---|---|
| Image formed by | Transmitted electrons | Scattered / secondary electrons from surface |
| Specimen type | Ultra‑thin sections or particles on grid | Bulk specimens |
| Typical resolution | 0.1–0.5 nm | 1–20 nm |
| Magnification range | Up to ×50 million | Up to ×500 000 |
| Image appearance | 2D projection, internal detail | 3D‑like surface topography |
| Depth of field | Low | Very high |
9. Advantages and limitations of electron microscopy | 电子显微镜的优点与局限
The most significant advantage of electron microscopy is its extraordinary resolution, which allows biologists to see structures right down to macromolecular complexes and individual atoms. Moreover, EM provides simultaneous visualisation of ultrastructure and context, especially when TEM is combined with tomography or SEM with correlative light‑electron microscopy. The ability to detect heavy‑atom labels (immunogold) also makes it possible to localise specific proteins within cells.
电子显微镜最显著的优点是其超凡的分辨率，使生物学家能够观察到直至生物大分子复合物和单个原子的结构。此外，电子显微镜能同时呈现超微结构及其环境背景，尤其是当透射电镜与断层成像技术结合，或扫描电镜与关联光‑电镜技术结合时。借助免疫金标记等重金属标签的检测能力，还能在细胞内对特定蛋白质进行定位。
However, electron microscopy has notable drawbacks. The vacuum environment and extensive preparation can introduce artefacts such as shrinkage, distortion or extraction of molecules. Living specimens cannot be observed — all samples are dead and fixed. The equipment is extremely expensive to purchase and maintain, and operation requires highly trained personnel. Moreover, the tiny field of view in TEM means that only a very small portion of a cell can be examined at ultra‑high resolution, which can lead to sampling bias.
然而，电子显微镜也存在明显不足。真空环境和复杂的制备过程可能引入人为假象，如收缩、变形或分子抽提。无法观察活体标本——所有样品都是死亡且被固定的。设备的购置与维护极其昂贵，且操作需要训练有素的人员。此外，透射电镜的视场极小，意味着只能以超高分辨率检查细胞的极小部分，这可能导致取样偏差。
10. Cryo‑electron microscopy (cryo‑EM) | 冷冻电子显微镜
Cryo‑EM has emerged as a revolutionary technique that sidesteps many of the artefacts associated with conventional EM preparation. Biological samples are rapidly frozen in vitreous ice, preserving them in a fully hydrated, near‑native state. No chemical fixatives or heavy‑metal stains are required, and the sample is imaged at cryogenic temperatures (below −150 °C). Single‑particle cryo‑EM allows the determination of three‑dimensional structures of macromolecules at resolutions that now routinely reach 2–3 Å, rivalling X‑ray crystallography without the need for crystals.
冷冻电镜已成为一项革命性技术，避免了许多与传统电镜制备相关的人为假象。生物样品在玻璃态冰中快速冷冻，以全水合、近天然状态加以保存。无需化学固定剂或重金属染色，样品在低温（低于零下150摄氏度）下成像。单颗粒冷冻电镜能够解析生物大分子的三维结构，分辨率现已常规达到2–3埃，可与X射线晶体学相媲美而无需晶体生长。
Recently, cryo‑electron tomography (cryo‑ET) has enabled visualisation of the molecular architecture inside intact cells or organelles, providing unprecedented views of cellular landscapes at molecular resolution. These developments collectively place electron microscopy at the forefront of modern structural and cell biology.
最近，冷冻电子断层扫描技术已能对完整细胞或细胞器内部的分子构造进行观察，在分子分辨率下呈现前所未有的细胞景观。这些发展共同将电子显微镜推向了现代结构生物学和细胞生物学的前沿。
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