📚 Electron Microscopy | 电子显微镜
Electron microscopy is a technique that uses a beam of accelerated electrons instead of visible light to examine specimens. In A-level biology, it is the key method for studying cell ultrastructure because it can resolve details far smaller than the wavelength of light. This article explains how electron microscopes work, compares the main types, and outlines their advantages, limitations and applications.
电子显微镜是一种利用加速电子束代替可见光来观察样品的技术。在 A-level 生物中，它是研究细胞超微结构的关键方法，因为它能分辨远小于光波长的细节。本文将解释电子显微镜的工作原理，比较主要类型，并概述其优势、局限和应用。
1. Principle of Electron Microscopy | 电子显微镜原理
An electron microscope uses a heated or field-emission electron gun to release electrons, which are accelerated by high voltage through a vacuum column. Electromagnetic lenses focus the beam; changing the current through these lenses alters magnification and focus.
电子显微镜使用热发射或场发射电子枪释放电子，电子在高电压下加速穿过真空镜筒。电磁透镜聚焦电子束；改变通过这些透镜的电流可调节放大倍数和聚焦。
e, typically 50-200 kV in a transmission electron microscope (TEM). The accelerated electrons carry kinetic energy equal to the work done by the accelerating voltage: eV = ½mₑv², where e = 1.60 × 10⁻¹⁹ C and mₑ = 9.11 × 10⁻³¹ kg. A higher voltage produces faster electrons and therefore a shorter electron wavelength.
电子由钨丝、六硼化镧（LaB₆）晶体或场发射枪释放，然后被高电压加速，在透射电镜（TEM）中通常为 50-200 kV。被加速的电子具有的动能等于加速电压所做的功：eV = ½mₑv²，其中 e = 1.60 × 10⁻¹⁹ C，mₑ = 9.11 × 10⁻³¹ kg。电压越高，电子越快，电子波长也越短。
eV = ½mₑv²   →   v = √2eV / mₑ
Because electrons are easily scattered by gas molecules, the specimen chamber and column must be kept under a high vacuum (about 10⁻⁵ Pa or better). This is one reason why living cells cannot be observed directly, and it also explains why the filament does not simply burn away in air.
由于电子很容易被气体分子散射，样品室和镜筒必须保持高真空（约 10⁻⁵ Pa 或更高）。这就是活细胞无法被直接观察的原因之一，也解释了为什么灯丝不会在空气中烧毁。
Instead of glass lenses, electron microscopes use electromagnetic coils that act as converging lenses. Changing the current through a coil changes its focal length, so the operator can adjust magnification and focus continuously. The final image is viewed on a fluorescent screen or captured by a CCD/CMOS camera as a digital greyscale image.
电子显微镜不用玻璃透镜，而用充当会聚透镜的电磁线圈。改变线圈中的电流会改变其焦距，因此操作者可以连续调节放大倍数与聚焦。最终图像在荧光屏上观察，或由 CCD/CMOS 相机记录为数字灰度图像。
2. Wavelength and Resolution | 波长与分辨率
Resolution is the ability to distinguish two points that are very close together. The smaller the resolvable distance, the higher the resolution. Resolution depends on the wavelength of the radiation used and on the quality of the lenses.
分辨率是区分两个靠得很近的点的能力。可分辨的距离越小，分辨率越高。分辨率取决于所用辐射的波长以及透镜的质量。
e far shorter: at an accelerating voltage of 100 kV, the de Broglie wavelength is only about 0.005 nm (5 × 10⁻¹² m), thousands of times shorter than light.
可见光的波长约为 400-700 nm，这使光学显微镜的分辨率被限制在约 200 nm。电子的波长要短得多：在 100 kV 加速电压下，德布罗意波长仅约 0.005 nm（5 × 10⁻¹² m），比光短数千倍。
λ = h / mv = h / √2mₑeV and  d = 0.61λ / (n sin θ)
d is the Rayleigh criterion, an approximation of the smallest distance d that can be resolved. Since λ is so small for electrons, the theoretical limit is extremely low; in practice, lens aberrations and specimen damage mean a TEM resolves about 0.1-0.2 nm and a SEM about 1-10 nm.
第一个式子是德布罗意方程，把波长与动量联系起来（h = 6.63 × 10⁻³⁴ J s）。第二个是瑞利判据，近似给出可分辨的最小距离 d。由于电子的 λ 极小，理论极限极低；但在实际中，透镜像差和样品损伤使 TEM 的分辨率约为 0.1-0.2 nm，SEM 约为 1-10 nm。
| Instrument | Resolution | Useful magnification |
| Human eye | ≈ 0.1 mm | ×1 |
| Optical (light) microscope | ≈ 200 nm | up to ×1500 |
| Scanning electron microscope (SEM) | 1-10 nm | ×10 to ×100 000 |
| Transmission electron microscope (TEM) | 0.1-0.2 nm | ×50 000 to ×1 000 000 |
Magnification and resolution are not the same thing. Magnification only enlarges an image, whereas resolution determines how much real detail can be seen. Beyond the resolution limit, further magnification produces empty magnification - a bigger but blurrier image with no extra detail.
放大倍数与分辨率不是一回事。放大只是把图像放大，而分辨率决定能看到多少真实细节。超过分辨率极限后继续放大只会得到空洞放大——图像更大却更模糊，没有增加任何细节。
3. Transmission Electron Microscope (TEM) | 透射电子显微镜（TEM）
In a TEM, the electron beam passes through an ultra-thin specimen (usually 50-100 nm thick) and the electrons that are transmitted are focused to form a two-dimensional image. Denser regions scatter more electrons and appear dark, so the image is a greyscale map of electron density.
在透射电镜中，电子束穿过超薄样品（通常厚 50-100 nm），透射过去的电子被聚焦成像，形成二维图像。较致密的区域散射更多电子而显得暗，因此图像是电子密度的灰度图。
Because electrons pass through the specimen, a TEM reveals internal structure: cristae inside mitochondria, the cisternae of rough and smooth endoplasmic reticulum, Golgi vesicles, nuclear pores, thylakoid grana in chloroplasts, ribosomes, microtubules, bacterial cell walls and the fine structure of viruses.
由于电子穿过样品，TEM 能揭示内部结构：线粒体嵴、粗面与光面内质网的扁囊、高尔基体囊泡、核孔、叶绿体类囊体基粒、核糖体、微管、细菌细胞壁以及病毒的精细结构。
Biological specimens are stained with heavy-metal salts such as uranyl acetate and lead citrate. These atoms scatter electrons strongly, so structures that bind the stain appear dark - this contrast has nothing to do with the natural colour of the specimen.
生物样品用乙酸铀、柠檬酸铅等重金属盐染色。这些原子强烈散射电子，因此结合染料的结构显得暗——这种对比与样品本身的颜色无关。
4. Scanning Electron Microscope (SEM) | 扫描电子显微镜（SEM）
In a SEM, the electron beam does not pass through the specimen. Instead, it is scanned across the surface point by point, and detectors collect the secondary electrons and back-scattered electrons that are emitted. The signal from each point is used to build up the image on a screen.
在扫描电镜中，电子束不穿过样品，而是逐点扫描表面，探测器收集发射出的二次电子和背散射电子。每个点的信号被用来在屏幕上构建图像。
Because the signal depends on the surface topography, the SEM image appears three-dimensional and shows surface features such as pollen grains, insect mouthparts, red blood cells, diatom shells, the surface of a leaf and biofilms on a catheter.
由于信号取决于表面形貌，SEM 图像具有立体感，显示表面特征，如花粉粒、昆虫口器、红细胞、硅藻外壳、叶片表面以及导管上的生物膜。
Specimens are usually fixed, dehydrated, dried by the critical-point method to avoid surface-tension artefacts, mounted on a stub and coated with a thin layer of gold or carbon so that the surface conducts charge away. Otherwise the specimen charges up and the image distorts.
样品通常经固定、脱水，用临界点干燥法避免表面张力造成的假象，再固定在样品台上并镀上一层薄金或碳，使表面能够导走电荷。否则样品会积累电荷，图像发生畸变。
5. Comparing TEM, SEM and the Light Microscope | TEM、SEM 与光学显微镜的比较
The three instruments answer different questions. The light microscope shows living cells and colour; the TEM shows internal ultrastructure at the highest resolution; the SEM shows surface topography in three dimensions.
三种仪器回答不同的问题。光学显微镜可观察活细胞并呈现颜色；TEM 以最高分辨率显示内部超微结构；SEM 以三维方式显示表面形貌。
| Feature | Light microscope | TEM | SEM |
| Radiation | visible light | electrons | electrons |
| Lenses | glass | electromagnetic | electromagnetic |
| Image type | 2D, coloured | 2D projection, greyscale | 3D-looking surface, greyscale |
| Living specimen | yes | no | no |
| Vacuum needed | no | yes | yes |
| Main use | cells, tissues, behaviour | organelles, viruses, molecules | surfaces, whole small organisms |
6. Specimen Preparation | 样品制备
Preparation is the step that most often decides whether an electron micrograph is trustworthy. Biological material must be preserved as close to its living state as possible, then made stable enough for the vacuum and the electron beam.
样品制备这一步往往决定一张电镜照片是否可信。生物材料必须尽可能接近生活状态被保存下来，然后被处理到足以承受真空和电子束。
- Primary fixation with glutaraldehyde cross-links proteins; secondary fixation with osmium tetroxide stabilises lipids and adds contrast. | 先用戊二醛初固定，使蛋白质交联；再用四氧化锇后固定，稳定脂类并增加对比。
- Dehydration through a graded ethanol or acetone series removes water. | 用梯度乙醇或丙酮系列脱水，除去水分。
- Embedding in resin allows the tissue to be cut. | 用树脂包埋，使组织能够被切片。
- Sectioning on an ultramicrotome produces sections 50-100 nm thick, supported on a copper grid. | 用超薄切片机切出 50-100 nm 厚的切片，置于铜网上。
- Staining with uranyl acetate and lead citrate creates electron contrast. | 用乙酸铀和柠檬酸铅染色，形成电子对比。
Modern alternatives include high-pressure freezing and cryo-electron microscopy (cryo-EM), in which specimens are frozen so rapidly that ice crystals do not form. Cryo-EM allows near-native structures to be imaged and was recognised by the 2017 Nobel Prize in Chemistry.
现代替代方法包括高压冷冻和冷冻电镜（cryo-EM），样品被极快速冷冻，从而不形成冰晶。冷冻电镜可对接近天然状态的结构成像，并因此获得 2017 年诺贝尔化学奖。
7. Advantages of Electron Microscopy | 电子显微镜的优势
The central advantage is resolution. Because the electron wavelength is thousands of times shorter than that of visible light, electron microscopes reveal organelles and macromolecular assemblies that are simply invisible in a light microscope.
核心优势是分辨率。由于电子波长比可见光短数千倍，电子显微镜能显示在光学显微镜中根本看不见的细胞器和大分子组装体。
- Very high resolution - down to about 0.1 nm in TEM, enough to see membranes, ribosomes and individual protein complexes. | 分辨率极高——TEM 可达约 0.1 nm，足以看见膜、核糖体和单个蛋白质复合体。
- Very high magnification - up to about ×1 000 000 with retained detail. | 放大倍数极高——可达约 ×1 000 000 且仍保留细节。
- Large depth of field, so structures at different depths stay in focus. | 景深大，不同深度的结构都能保持清晰。
- SEM gives an almost 3D view of surfaces. | SEM 提供近乎三维的表面视图。
- Digital images can be measured, annotated and shared, and the scale bar allows real sizes to be calculated. | 数字图像可测量、标注和共享，比例尺可用于计算真实尺寸。
- Coupling with analytical detectors (EDX) gives the elemental composition of a region of the specimen. | 与分析型探测器（EDX）联用，可给出样品某一区域的元素组成。
- Immunogold labelling uses gold particles attached to antibodies to pinpoint the location of a specific protein. | 免疫金标记用连接抗体的金颗粒定位特定蛋白质。
8. Limitations of Electron Microscopy | 电子显微镜的局限
The same physics that gives the electron microscope its power also creates its restrictions. Electrons travel only in a vacuum and interact strongly with matter, so specimens must be dead, dry and very stable.
赋予电子显微镜强大能力的物理原理同时也带来了限制。电子只能在真空中传播，并与物质强烈作用，因此样品必须是死的、干燥的并且非常稳定。
- Living specimens cannot be viewed, so dynamic processes cannot be followed directly. | 无法观察活样品，因此不能直接追踪动态过程。
- The specimen must be in a high vacuum, which removes water and gases. | 样品必须处于高真空，这会除去水分和气体。
- Extensive preparation can introduce artefacts such as shrinkage or distortion. | 繁复的制备可能引入皱缩或变形等假象。
- The electron beam can damage the specimen, especially at high magnification. | 电子束可能损伤样品，高倍下尤为明显。
- Images are greyscale; colour must be added artificially for display. | 图像是灰度的；用于展示的颜色是人工添加的。
- TEM specimens must be ultra-thin, so only a small volume is examined, and a TEM image is a 2D projection of a 3D object. | TEM 样品必须超薄，因此只检查很小体积，且 TEM 图像是三维物体的二维投影。
- Equipment is expensive to buy, run and maintain, and requires trained staff and specialised preparation facilities. | 设备购置、运行和维护成本高，需要专业人员与专门的制备设施。
- There is no colour information about biological stains as in a light micrograph. | 不像光学显微照片那样能提供生物染色的颜色信息。
9. Artefacts and Image Interpretation | 假象与图像解读
An artefact is a feature seen in the image that was produced by preparation or imaging rather than existing in the living specimen. Common artefacts include shrinkage during dehydration, knife marks from sectioning, stain precipitate, folds in the section and holes where ice crystals formed.
假象是指在图像中看到、但由制备或成像过程产生而非活体样品中真实存在的特征。常见假象包括脱水造成的皱缩、切片留下的刀痕、染色沉淀、切片折叠以及冰晶形成的空洞。
When interpreting an electron micrograph, always use the scale bar to estimate real sizes, then match those sizes to known structures. Typical values worth remembering are shown below.
解读电镜照片时，先用比例尺估算真实尺寸，再把尺寸与已知结构对应。下表列出值得记住的典型数值。
| Structure | Approximate size |
| Cell-surface membrane thickness | 7 nm |
| Ribosome diameter | 20-25 nm |
| Mitochondrion length | 1-10 µm |
| Chloroplast length | 5-10 µm |
| Bacterium diameter | 0.5-5 µm |
| Typical animal cell | 10-30 µm |
s actual size = image size ÷ magnification, which is exactly what a scale bar lets you do.
一个常用计算是放大倍数：放大倍数 = 图像大小 ÷ 物体实际大小。变形可得：实际大小 = 图像大小 ÷ 放大倍数，这正是比例尺能帮我们完成的计算。
magnification = image size ÷ actual size
10. Applications in Biology and Beyond | 在生物学及其他领域的应用
Electron microscopy underpins much of modern cell biology and has spread into medicine, materials science and nanotechnology.
电子显微镜支撑了现代细胞生物学的许多内容，并已扩展到医学、材料科学和纳米技术。
- Mapping cell ultrastructure, including the endomembrane system, cytoskeleton and cell junctions. | 绘制细胞超微结构，包括内膜系统、细胞骨架和细胞连接。
- Identifying viruses and observing their structure, for example the spike proteins of coronaviruses. | 鉴定病毒并观察其结构，例如冠状病毒的刺突蛋白。
- Diagnosing disease in biopsy samples, such as kidney disorders caused by abnormal basement membranes. | 通过活检样品诊断疾病，如基底膜异常引起的肾脏疾病。
- Determining protein structures by cryo-EM, complementing X-ray crystallography. | 用冷冻电镜测定蛋白质结构，与 X 射线晶体学互补。
- Studying bacterial flagella, pili and biofilm formation. | 研究细菌鞭毛、菌毛和生物膜形成。
- Examining surfaces in materials science, semiconductor manufacture and forensic analysis of trace evidence. | 在材料科学、半导体制造和痕量物证的法庭鉴定中检查表面。
11. Exam Skills and Command Words | 考试技巧与答题要点
Examiners reward precise
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