When light passes through a living cell, the phase of the light wave is changed according to the cell’s refractive index: a relatively thick or dense part of the cell, such as a nucleus, slows the light passing through it. The phase of the light, consequently, is shifted relative to light that has passed through an adjacent thinner region of the cytoplasm (Figure 9–6C). The phase-contrast microscope and, in a more complex way, the differential-interference-contrast microscope increase these phase differences to produce amplitude differences, or contrast, when the sets of waves recombine, thereby creating an image of the cell’s structure.

当光穿过一个活细胞时，光波的相位会依细胞的折射率而改变：细胞中较厚或较致密的部位（例如细胞核）会使穿过它的光减慢。因此，相对于穿过邻近较薄的细胞质区域的光，此处光的相位发生了位移（图9–6C）。相差显微镜，以及以更复杂方式实现同样目的的微分干涉差显微镜，会放大这些相位差，使两组光波重新汇合时转变为振幅差、即反差，从而形成细胞结构的图像。

This microscope is similar to an ordinary upright or inverted light microscope except that the illuminating light, from a very powerful source, is passed through two sets of filters—one to filter the light before it reaches the specimen, and one to filter the light obtained from the specimen. The first filter passes only the wavelengths that excite the particular fluorescent dye, while the second filter blocks out this light and passes only those wavelengths emitted when the dye fluoresces (Figure 9–10C). Fluorescence microscopy is most often used to detect specific proteins or other molecules in cells and tissues.

这种显微镜与普通的正置或倒置光学显微镜相似，区别在于来自极强光源的照明光要通过两组滤光片——一组在光到达标本之前滤光，另一组滤过来自标本的光。第一片滤光片只让能激发该特定荧光染料的波长通过，第二片则挡掉这些激发光、只放行染料发出荧光时的发射波长（图9–10C）。荧光显微术最常用于检测细胞和组织中的特定蛋白质或其他分子。

The precise antigen specificity of antibodies makes them powerful tools for the cell biologist. When chemically coupled to fluorescent dyes, antibodies are invaluable for locating specific molecules in cells by fluorescence microscopy (Figure 9–14). When labeled with electron-dense particles such as colloidal gold spheres, they are used for similar purposes in the electron microscope (discussed later). The antibodies employed in microscopy are commonly either purified from antiserum so as to remove all nonspecific antibodies or they are specific monoclonal antibodies that only recognize the target molecule.

抗体精确的抗原特异性使它们成为细胞生物学家的有力工具。当与荧光染料化学偶联后，抗体在荧光显微镜下定位细胞内特定分子时极为有用（图9–14）。当用胶体金球等电子致密颗粒标记时，它们在电子显微镜下用于同样的目的（后文讨论）。显微术中所用的抗体，通常要么是从抗血清中纯化、除去了全部非特异性抗体的抗体，要么是只识别靶分子的特异性单克隆抗体。

Because the specimen is exposed to a very high vacuum in the electron microscope, living tissue is usually killed and preserved by chemical fixation. As electrons have very limited penetrating power, the fixed tissues normally have to be cut into extremely thin sections (25–100 nm thick, about 1/200 the thickness of a single cell) before they are viewed. This is achieved by dehydrating the specimen, permeating it with a monomeric resin that polymerizes to form a solid block of plastic, then cutting the block with a fine glass or diamond knife on a special microtome.

由于标本在电子显微镜中要处于极高真空环境，活组织通常先被杀死并用化学固定加以保存。因为电子的穿透能力非常有限，固定后的组织通常必须切成极薄的切片（厚 25–100 nm，约为单个细胞厚度的 1/200）才能观察。做法是先对标本脱水，再用单体树脂浸透，树脂聚合后形成固体塑料块，然后在专门的切片机上用锋利的玻璃刀或金刚石刀切开该块。

Some rapidly frozen specimens can be examined directly in the electron microscope using a special cooled specimen holder. In other cases, the frozen block can be fractured to reveal interior cell surfaces or the surrounding ice can be sublimed away to expose external surfaces. However, we often want to examine thin sections, and the frozen tissue can be sectioned directly in a cooled microtome.

有些快速冷冻的标本可以用特制的冷冻样品台直接在电子显微镜中观察。在另一些情况下，可以把冷冻的样品块断裂开以暴露细胞内部的断面，或者让周围的冰升华掉以暴露外表面。不过我们常常还是希望观察薄切片，此时冷冻组织可以直接在低温切片机上切片。

Such cell fractionations became possible only after the commercial development in the early 1940s of an instrument known as the preparative ultracentrifuge, which rotates extracts of broken cells at high speeds (Figure 8–4). This treatment separates cell components by size and density: in general, the largest objects experience the largest centrifugal force and move the most rapidly. At relatively low speed, large components such as nuclei sediment to form a pellet at the bottom of the centrifuge tube; at slightly higher speed, a pellet of mitochondria is deposited; and at even higher speeds and with longer periods of centrifugation, first vesicles and then ribosomes can be collected (Figure 8–5).

这类细胞分级分离只有在 20 世纪 40 年代初制备型超速离心机商品化之后才成为可能，该仪器把破碎细胞的提取物高速旋转（图8–4）。这种处理按大小和密度分离细胞组分：一般来说，最大的颗粒受到的离心力最大、移动最快。在相对低速时，细胞核这类大组分沉降到离心管底部形成沉淀；转速略高时线粒体沉淀下来；在更高转速和更长的离心时间下，先是囊泡、随后是核糖体被收集下来（图8–5）。

Most vertebrate cells stop dividing after a finite number of cell divisions in culture, a process called replicative cell senescence (discussed in Chapter 17). Normal human fibroblasts, for example, typically divide only 25–40 times in culture before they stop. In these cells, the limited proliferation capacity reflects a progressive shortening and uncapping of the cell’s telomeres, the repetitive DNA sequences and associated proteins that cap the ends of each chromosome (discussed in Chapter 5).

大多数脊椎动物细胞在培养中分裂有限次数之后就停止分裂，这一过程称为复制性细胞衰老（见第17章）。例如正常人成纤维细胞在培养中通常只分裂 25–40 次就停下来。在这些细胞中，增殖能力受限反映的是细胞端粒的逐渐缩短与"去帽"，端粒即封盖每条染色体末端的重复 DNA 序列及其结合蛋白（见第5章）。

First, interactions between one protein and another can be monitored by Förster resonance energy transfer, also called fluorescence resonance energy transfer but both abbreviated to FRET. In this technique, two molecules of interest are each labeled with a different fluorochrome, chosen so that the emission spectrum of one fluorochrome, the donor, overlaps with the absorption spectrum of the other, the acceptor. If the two proteins interact in such a way as to bring their fluorochromes into very close proximity (closer than about 5 nm), one fluorochrome, when excited, can transfer energy from the absorbed light directly (by resonance, nonradiatively) to the other.

首先，蛋白质之间的相互作用可以用福斯特共振能量转移来监测，它也叫荧光共振能量转移，两者都缩写为 FRET。在该技术中，两个目标分子各自用一种不同的荧光基团标记，选择的原则是：其中一个荧光基团（供体）的发射光谱与另一个（受体）的吸收光谱相互重叠。如果两个蛋白质发生相互作用、使它们的荧光基团靠得非常近（近于约 5 nm），那么被激发的那个荧光基团就能把吸收的光能直接地（以共振方式、非辐射地）转移给另一个。

This method, called equilibrium sedimentation, is so sensitive that it can separate macromolecules that have incorporated heavy isotopes, such as 13C or 15N, from the same macromolecules that contain the lighter, common isotopes (12C or 14N). In fact, the cesium-chloride method was developed in 1957 to separate the labeled from the unlabeled DNA produced after exposure of a growing population of bacteria to nucleotide precursors containing 15N; this classic experiment provided direct evidence for the semiconservative replication of DNA (see Figure 5–5).

这种方法称为平衡沉降，它非常灵敏，能把掺入了 13C 或 15N 等重同位素的大分子，与含较轻常见同位素（12C 或 14N）的同种大分子分开。事实上，氯化铯法正是在 1957 年为分离细菌在含 15N 的核苷酸前体中生长后产生的标记 DNA 与未标记 DNA 而建立的；这一经典实验为 DNA 的半保留复制提供了直接证据（见图5–5）。

Another way to exploit GFP fused to a protein of interest is known as fluorescence recovery after photobleaching (FRAP). Here, a strong focused beam of light from a laser is used to extinguish the GFP fluorescence in a specified region of the cell, after which one can analyze the way in which remaining unbleached fluorescent protein molecules move into the bleached area as a function of time. This technique, like photoactivation, can deliver valuable quantitative data about a protein’s kinetic parameters, such as diffusion coefficients, active transport rates, or binding and dissociation rates from other proteins (Figure 9–20).

利用与目标蛋白融合的 GFP 的另一种方式，称为光漂白后荧光恢复（FRAP）。在这里，用来自激光的强聚焦光束把细胞中某一指定区域内的 GFP 荧光淬灭，随后就可以分析剩余未被漂白的荧光蛋白分子如何随时间迁入被漂白的区域。这项技术与光激活一样，能提供关于某蛋白动力学参数的宝贵定量数据，例如扩散系数、主动运输速率，或与其他蛋白的结合与解离速率（图9–20）。

Molecules in all kinds of thin sections can be labeled to identify and localize them. We have seen earlier how antibodies can be used in conjunction with fluorescence microscopy to localize specific macromolecules. An analogous method—immunogold electron microscopy—can be used in the electron microscope. The usual procedure is to incubate a thin section first with a specific primary antibody, and then with a secondary antibody to which a colloidal gold particle has been attached. The gold particle is electron-dense and can be seen as a black dot in the electron microscope (Figure 9–43).

各种薄切片中的分子都可以被标记，以便鉴定和定位。前面我们已经看到抗体如何与荧光显微术结合来定位特定的大分子。一种与之对应的方法——免疫金电子显微术——可以用于电子显微镜。通常的做法是先用特异性一抗孵育薄切片，再用连有胶体金颗粒的二抗孵育。金颗粒是电子致密的，在电子显微镜下可见为一个黑点（图9–43）。

Because the aberrations of an electron lens are considerably harder to correct than those of a glass lens, however, the practical resolving power of modern electron microscopes is, even with careful image processing to correct for lens aberrations, about 0.05 nm (0.5 Å) (Figure 9–39). This is because only the very center of the electron lenses can be used, and the effective numerical aperture is tiny. Furthermore, problems of specimen preparation, contrast, and radiation damage have generally limited the normal effective resolution for biological objects to 1 nm (10 Å). This is nonetheless about 200 times better than the resolution of the light microscope.

然而，由于电子透镜的像差比玻璃透镜的像差难校正得多，即使配合仔细的图像处理来校正透镜像差，现代电子显微镜实际的分辨本领也只有约 0.05 nm（0.5 Å）（图9–39）。这是因为只能使用电子透镜最中心的部分，有效数值孔径极小。此外，制样、反差和辐射损伤等问题，通常把生物样品的常规有效分辨率限制在 1 nm（10 Å）。即便如此，这仍比光学显微镜的分辨率高约 200 倍。

A specific protein can be identified after its fractionation on a polyacrylamide gel by exposing all the proteins on the gel to a specific antibody that has been labeled with a radioactive isotope or a fluorescent dye. This procedure is normally carried out after transferring all of the separated proteins in the gel onto a sheet of nitrocellulose paper or nylon membrane. Placing the membrane against the gel and driving the proteins out of the gel with a strong electric current transfers the protein onto the membrane. The membrane is then soaked in a solution of labeled antibody to reveal the protein of interest. This method of detecting proteins is called Western blotting, or immunoblotting (Figure 8–17).

一种特定蛋白质在聚丙烯酰胺凝胶上分离之后，可以通过把凝胶上的全部蛋白质暴露于一种用放射性同位素或荧光染料标记的特异性抗体来加以鉴定。这一步通常是在把凝胶中已分离的全部蛋白质转移到硝酸纤维素膜或尼龙膜上之后进行的。把膜贴在凝胶上，用强电流把蛋白质从凝胶中驱出，即可把蛋白质转移到膜上。然后把膜浸在标记抗体的溶液中，从而显示出目标蛋白。这种检测蛋白质的方法称为 Western 印迹法，或免疫印迹法（图8–17）。

Another optical method for probing protein interactions uses green fluorescent protein (discussed in detail later) and its derivatives of different colors. In this application, two proteins of interest are each labeled with a different fluorescent protein, such that the emission spectrum of one fluorescent protein overlaps the absorption spectrum of the second. If the two proteins come very close to each other (within about 1–5 nm), the energy of the absorbed light is transferred from one fluorescent protein to the other. The energy transfer, called fluorescence resonance energy transfer (FRET), is determined by illuminating the first fluorescent protein and measuring emission from the second (see Figure 9–19).

另一种探测蛋白质相互作用的光学方法利用绿色荧光蛋白（后文详述）及其不同颜色的衍生物。在这一应用中，两个目标蛋白各自用一种不同的荧光蛋白标记，使其中一种荧光蛋白的发射光谱与另一种的吸收光谱相重叠。如果两个蛋白彼此靠得非常近（约 1–5 nm 之内），被吸收的光能就会从一种荧光蛋白转移到另一种。这种能量转移称为荧光共振能量转移（FRET），其判定方式是照射第一种荧光蛋白并测量第二种的发射（见图9–19）。

This fundamental capacity of a single-strand nucleic acid molecule, either DNA or RNA, to form a double helix with a single-strand molecule of a complementary sequence provides a powerful and sensitive technique for detecting specific nucleotide sequences. Today, one simply designs a short, single-strand DNA molecule (often called a DNA probe) that is complementary to the nucleotide sequence of interest. Because the nucleotide sequences of so many genomes are known—and are stored in publicly accessible databases—designing a probe to hybridize anywhere in a genome is straightforward.

单链核酸分子（DNA 或 RNA 均可）能与具互补序列的单链分子形成双螺旋，这一基本能力提供了检测特定核苷酸序列的强大而灵敏的技术。今天，人们只需设计一段与目标核苷酸序列互补的短单链 DNA 分子（常称为 DNA 探针）。由于如此多基因组的核苷酸序列都已知晓、并存放在可公开检索的数据库中，设计一条能杂交到基因组任意位置的探针是很容易的事。

A key method for identifying proteins that bind to one another tightly is co-immunoprecipitation. A target protein is immunoprecipitated from a cell lysate using specific antibodies coupled to beads, as described earlier. If the target protein is associated tightly enough with another protein when it is captured by the antibody, the partner precipitates as well and can be identified by mass spectrometry. This method is useful for identifying proteins that are part of a complex inside cells, including those that interact only transiently; for example, when extracellular signal molecules stimulate cells (discussed in Chapter 15).

鉴定彼此紧密结合的蛋白质，一个关键方法是免疫共沉淀。用偶联到珠子上的特异性抗体，把靶蛋白从细胞裂解液中免疫沉淀下来（如前所述）。如果靶蛋白被抗体捕获时与另一种蛋白结合得足够紧密，那么这个伙伴蛋白也会一并被沉淀下来，并可用质谱加以鉴定。该方法适用于鉴定细胞内属于同一复合物的蛋白，包括那些只发生瞬时相互作用的蛋白；例如细胞外信号分子刺激细胞时的情形。

Sometimes, a double mutant will show a new or more severe phenotype than either single mutant alone. This type of genetic interaction is called a synthetic phenotype, and if the phenotype is death of the organism, it is called synthetic lethality. In most cases, a synthetic phenotype indicates that the two genes act in two different parallel pathways, either of which is capable of mediating the same cell process. Thus, when both pathways are disrupted in the double mutant, the process fails altogether, and the synthetic phenotype is observed.

有时，双突变体会表现出比任一单突变体都新的或更严重的表型。这类遗传相互作用称为合成表型；如果该表型是生物体死亡，就称为合成致死。多数情况下，出现合成表型说明这两个基因作用于两条不同的平行通路上，而其中任一条都能介导同一细胞过程。因此，当双突变体把两条通路都破坏掉时，该过程就彻底失败，于是观察到合成表型。

B lymphocytes normally have a limited life span in culture, but individual antibody-producing B lymphocytes from an immunized mouse, when fused with cells derived from a transformed B lymphocyte cell line, can give rise to hybrids that have both the ability to make a particular antibody and the ability to multiply indefinitely in culture (Figure 8–3). These hybridomas are propagated as individual clones, each of which provides a permanent and stable source of a single type of monoclonal antibody. Each type of monoclonal antibody recognizes a single type of antigenic site; for example, a particular cluster of five or six amino acid side chains on the surface of a protein.

B 淋巴细胞在培养中通常寿命有限，但来自免疫小鼠的单个产抗体 B 淋巴细胞，与源自转化 B 淋巴细胞系的细胞融合后，可以产生同时具备两种能力的杂种细胞：既能制造某一特定抗体，又能在培养中无限增殖（图8–3）。这些杂交瘤以单个克隆的形式扩增，每个克隆都提供一种单一类型单克隆抗体的永久而稳定的来源。每一种单克隆抗体只识别一种类型的抗原位点；例如蛋白表面某一簇由五六个氨基酸侧链组成的位点。

Negative staining is a similar approach that also allows fine detail to be seen in isolated molecules or macromolecular machines. In this technique, the molecules are supported on the thin film of carbon on a grid and mixed with a solution of a heavy-metal salt such as uranyl formate or acetate. After the sample has dried, a very thin film of metal salt covers the carbon film everywhere except where it has been excluded by the presence of an adsorbed macromolecule.

负染色是一种类似的方法，同样能显示分离的分子或大分子机器的精细结构。在该技术中，分子被支撑在载网的碳支持膜上，并与重金属盐溶液（如甲酸铀酰或醋酸铀酰）混合。样品干燥后，除被吸附的大分子占据（把染液排开）的位置以外，碳膜各处都覆盖上一层极薄的金属盐薄膜。

The shapes of isolated molecules can be roughly determined by electron microscopy techniques involving negative staining or heavy-metal shadowing, but detailed molecular structures require cryoEM and single-particle reconstruction using computational manipulations of data obtained from multiple images and multiple viewing angles to produce detailed reconstructions of macromolecules and molecular complexes.

分离出来的分子，其形状可以用负染色或重金属投影这类电镜技术大致测定；但精细的分子结构则需要冷冻电镜和单颗粒重构——即对多幅图像、多个观察角度所得的数据进行计算处理，从而重建出大分子及大分子复合物的精细结构。

The cell cultures obtained by disrupting tissues tend to suffer from a problem—eventually the cells die. Most vertebrate cells stop dividing after a finite number of cell divisions in culture, a process called replicative cell senescence (discussed in Chapter 17). Normal human fibroblasts, for example, typically divide only 25–40 times in culture before they stop.

通过打散组织得到的细胞培养物往往有一个问题——细胞最终会死亡。多数脊椎动物细胞在培养中经过有限次数的分裂后就停止分裂，这一过程称为复制性细胞衰老（见第 17 章）。例如，正常人成纤维细胞在培养中通常只分裂 25～40 次就停下来。

Many animal and plant cells survive and proliferate in a culture dish if they are provided with a suitable culture medium containing nutrients and appropriate signal molecules. Although many animal cells stop dividing after a finite number of cell divisions, cells that have been immortalized through spontaneous mutations or genetic manipulation can be maintained indefinitely as cell lines.

只要提供含有营养物和适当信号分子的合适培养基，许多动物细胞和植物细胞都能在培养皿中存活并增殖。虽然许多动物细胞在有限次数的分裂后就停止分裂，但那些通过自发突变或遗传操作而实现永生化的细胞，可以作为细胞系被无限期地维持下去。

For example, the experiments to decipher the mechanisms of protein synthesis began with a cell homogenate that could translate RNA molecules to produce proteins. Fractionation of this homogenate, step by step, produced in turn the ribosomes, tRNAs, and various enzymes that together constitute the protein-synthetic machinery. Once individual pure components were available, each could be added or withheld separately to define its exact role in the overall process.

例如，破译蛋白质合成机制的那些实验，是从一份能够翻译 RNA 分子以生成蛋白质的细胞匀浆开始的。对这份匀浆逐步进行分级分离，依次得到了核糖体、tRNA 以及共同构成蛋白质合成机器的各种酶。一旦获得了单个的纯化组分，就可以逐一加入或去除，以确定它在整个过程中的确切作用。

A major goal for cell biologists is the reconstitution of every biological process in a purified cell-free system. Only in this way can we define all of the components needed for the process and control their concentrations, which is required to work out their precise mechanism of action. Although much remains to be done, a great deal of what we know today about the molecular biology of the cell has been discovered by studies in such cell-free systems. They have been used, for example, to decipher the molecular details of DNA replication and DNA transcription, RNA splicing, protein translation, muscle contraction, particle transport along microtubules, and many other processes that occur in cells.

细胞生物学家的一个主要目标，是在纯化的非细胞体系中重建每一个生物学过程。只有这样，我们才能确定该过程所需的全部组分并控制它们的浓度，而这正是弄清其精确作用机制所必需的。尽管仍有大量工作要做，但我们今天所掌握的细胞分子生物学知识，有很大一部分正是通过这类非细胞体系的研究获得的。例如，它们已被用于阐明 DNA 复制与 DNA 转录、RNA 剪接、蛋白质翻译、肌肉收缩、颗粒沿微管运输，以及细胞内许多其他过程的分子细节。

Because the macromolecule allows electrons to pass through it much more readily than does the surrounding heavy-metal stain, a reverse or negative image of the molecule is created. Negative staining is especially useful for quickly and cheaply viewing large macromolecular aggregates such as viruses or ribosomes and for seeing the subunit structure of protein filaments (Figure 9–44B). Shadowing and negative staining can provide high-contrast surface views of small macromolecular assemblies, but the size of the smallest metal particles in the shadow or stain limits the resolution of both techniques to about 2 nm.

由于电子穿过大分子远比穿过周围的重金属染液容易，于是形成该分子的一个反转的、即负性的图像。负染色特别适合快速而廉价地观察病毒、核糖体这类大型大分子聚合体，也适合观察蛋白质纤维的亚基结构（图 9–44B）。投影（喷镀）与负染色都能给出小型大分子组装体的高反差表面形貌，但阴影或染液中最小金属颗粒的尺寸，把这两种技术的分辨率限制在约 2 nm。

A special sample holder keeps this hydrated specimen at –160°C in the vacuum of the microscope, where it can be viewed directly without fixation, staining, or drying. Unlike negative staining, in which what we see is the envelope of stain exclusion around the particle, cryoEM produces an image from the macromolecular structure itself.

特制的样品台把这一含水样品保持在显微镜真空环境中的 –160°C，使其无需固定、染色或干燥即可直接观察。与负染色不同——负染色看到的是颗粒周围染液被排开所形成的外廓——冷冻电镜得到的图像来自大分子结构本身。

Provided that the homogenization medium has been carefully chosen (by trial and error for each organelle), the various components—including the vesicles derived from the endoplasmic reticulum, called microsomes—retain most of their original biochemical properties. The different components of the homogenate must then be separated. Such cell fractionations became possible only after the commercial development in the early 1940s of an instrument known as the preparative ultracentrifuge, which rotates extracts of broken cells at high speeds (Figure 8–4).

只要匀浆介质选择得当（针对每种细胞器反复试验确定），各种组分——包括由内质网衍生而来、被称为微粒体的那些囊泡——都能保留其大部分原有的生化特性。随后必须把匀浆中的不同组分分开。这类细胞分级分离只有在 20 世纪 40 年代初制备型超速离心机作为商品问世之后才成为可能，该仪器可使破碎细胞的提取物高速旋转（图 8–4）。

These procedures break many of the membranes of the cell (including the plasma membrane and endoplasmic reticulum) into fragments that immediately reseal to form small closed vesicles. If carefully carried out, however, the disruption procedures leave organelles such as nuclei, mitochondria, the Golgi apparatus, lysosomes, and peroxisomes largely intact.

这些操作把细胞的许多膜（包括质膜和内质网）打碎成片段，这些片段随即重新封闭，形成小的封闭囊泡。但若操作得当，破碎过程仍会使细胞核、线粒体、高尔基体、溶酶体和过氧化物酶体等细胞器基本保持完整。

Studies of organelles and other large subcellular components isolated in the ultracentrifuge have contributed enormously to our understanding of the functions of different cell components. Experiments on mitochondria and chloroplasts purified by centrifugation, for example, demonstrated the central function of these organelles in converting energy into forms that the cell can use. Similarly, resealed vesicles formed from fragments of rough and smooth endoplasmic reticulum (microsomes) have been separated from each other and analyzed as functional models of these compartments of the intact cell.

用超速离心分离得到的细胞器及其他大的亚细胞组分，对它们的研究极大地增进了我们对各种细胞组分功能的理解。例如，用离心纯化的线粒体和叶绿体所做的实验，证明了这些细胞器在把能量转换成细胞可利用形式方面的核心作用。同样，由粗面内质网与光面内质网片段重新封闭形成的囊泡（即微粒体）也已被彼此分开，并作为完整细胞中这些区室的功能模型加以分析。

Another way to exploit GFP fused to a protein of interest is known as fluorescence recovery after photobleaching (FRAP). Here, a strong focused beam of light from a laser is used to extinguish the GFP fluorescence in a specified region of the cell, after which one can analyze the way in which remaining unbleached fluorescent protein molecules move into the bleached area as a function of time. This technique, like photoactivation, can deliver valuable quantitative data about a protein’s kinetic parameters, such as diffusion coefficients, active transport rates, or binding and dissociation rates from other proteins (Figure 9–20).

利用与目的蛋白融合的 GFP 的另一种做法，称为光漂白后荧光恢复（FRAP）。其做法是：用激光发出的强聚焦光束熄灭细胞某一指定区域内的 GFP 荧光，随后即可分析剩余未被漂白的荧光蛋白分子随时间迁入漂白区域的方式。该技术与光激活一样，能给出关于蛋白质动力学参数的有价值的定量数据，例如扩散系数、主动运输速率，或与其他蛋白质的结合与解离速率（图 9–20）。

A strong focused pulse of laser light will extinguish, or bleach, fluorescent proteins. By selectively photobleaching a set of fluorescently tagged protein molecules within a defined region of a cell, the microscopist can monitor recovery over time, as the remaining fluorescent molecules move into the bleached region (see Movie 10.6).

一束强聚焦的激光脉冲会熄灭（即漂白）荧光蛋白。通过选择性地漂白细胞内某一限定区域中的一组荧光标记蛋白质分子，显微镜使用者就能随时间监测荧光的恢复，因为其余未被漂白的荧光分子会不断迁入该漂白区域（见 Movie 10.6）。

Another way to exploit GFP fused to a protein of interest is known as fluorescence recovery after photobleaching (FRAP). Here, a strong focused beam of light from a laser is used to extinguish the GFP fluorescence in a specified region of the cell, after which one can analyze the way in which remaining unbleached fluorescent protein molecules move into the bleached area as a function of time. This technique, like photoactivation, can deliver valuable quantitative data about a protein’s kinetic parameters, such as diffusion coefficients, active transport rates, or binding and dissociation rates from other proteins (Figure 9–20).

利用与目的蛋白融合的 GFP 的另一种做法，称为光漂白后荧光恢复（FRAP）。其做法是：用激光发出的强聚焦光束熄灭细胞某一指定区域内的 GFP 荧光，随后即可分析剩余未被漂白的荧光蛋白分子随时间迁入漂白区域的方式。该技术与光激活一样，能给出关于蛋白质动力学参数的有价值的定量数据，例如扩散系数、主动运输速率，或与其他蛋白质的结合与解离速率（图 9–20）。

A strong focused pulse of laser light will extinguish, or bleach, fluorescent proteins. By selectively photobleaching a set of fluorescently tagged protein molecules within a defined region of a cell, the microscopist can monitor recovery over time, as the remaining fluorescent molecules move into the bleached region (see Movie 10.6).

一束强聚焦的激光脉冲会熄灭（即漂白）荧光蛋白。通过选择性地漂白细胞内某一限定区域中的一组荧光标记蛋白质分子，显微镜使用者就能随时间监测荧光的恢复，因为其余未被漂白的荧光分子会不断迁入该漂白区域（见 Movie 10.6）。

B lymphocytes normally have a limited life span in culture, but individual antibody-producing B lymphocytes from an immunized mouse, when fused with cells derived from a transformed B lymphocyte cell line, can give rise to hybrids that have both the ability to make a particular antibody and the ability to multiply indefinitely in culture (Figure 8–3). These hybridomas are propagated as individual clones, each of which provides a permanent and stable source of a single type of monoclonal antibody.

B 淋巴细胞在培养中通常寿命有限，但取自免疫小鼠的单个产抗体 B 淋巴细胞，在与来源于转化 B 淋巴细胞系的细胞融合后，可以产生既能制造某一特定抗体、又能在培养中无限增殖的杂交细胞（图 8–3）。这些杂交瘤被作为一个个单独的克隆加以扩增，每个克隆都提供某一单一类型单克隆抗体的永久而稳定的来源。

But if the B lymphocytes that produce the various components of this antiserum are made into hybridomas, it becomes possible to screen individual hybridoma clones from the large mixture to select one that produces the desired type of monoclonal antibody and to propagate the selected hybridoma indefinitely so as to produce that antibody in unlimited quantities.

但如果把产生这份抗血清中各种成分的 B 淋巴细胞制成杂交瘤，就有可能从庞大的混合群体中筛选单个杂交瘤克隆，挑出能产生所需类型单克隆抗体的那一个，并把选中的杂交瘤无限扩增，从而无限量地生产该抗体。

Cell lines can often be most easily generated from cancer cells, but these cultures—referred to as transformed cell lines—differ from those prepared from normal cells in several ways. Transformed cell lines often grow without attaching to a surface, for example, and they can proliferate to a much higher density in a culture dish. Similar properties can be induced experimentally in normal cells by transforming them with a tumor-inducing virus or chemical.

细胞系往往最容易由癌细胞建立，但这类培养物——被称为转化细胞系——在若干方面不同于由正常细胞制备的培养物。例如，转化细胞系常常不贴附于表面也能生长，而且能在培养皿中增殖到高得多的密度。用致瘤病毒或化学致癌剂转化正常细胞，也可以在实验上诱导出类似的性质。

First, interactions between one protein and another can be monitored by Förster resonance energy transfer, also called fluorescence resonance energy transfer but both abbreviated to FRET. In this technique, two molecules of interest are each labeled with a different fluorochrome, chosen so that the emission spectrum of one fluorochrome, the donor, overlaps with the absorption spectrum of the other, the acceptor. If the two proteins interact in such a way as to bring their fluorochromes into very close proximity (closer than about 5 nm), one fluorochrome, when excited, can transfer energy from the absorbed light directly (by resonance, nonradiatively) to the other.

首先，一种蛋白质与另一种蛋白质之间的相互作用，可以通过福斯特共振能量转移来监测，它也被称为荧光共振能量转移，两者都缩写为 FRET。在该技术中，两个目标分子各自用一种不同的荧光基团标记，所选的荧光基团须满足：其中一个（供体）的发射光谱与另一个（受体）的吸收光谱相互重叠。如果两种蛋白质的相互作用使它们的荧光基团彼此靠得非常近（近于约 5 nm），那么其中一个荧光基团被激发后，就能把所吸收光的能量直接（以共振方式、非辐射地）转移给另一个。

Antibodies are proteins produced by the vertebrate immune system as a defense against infection (discussed in Chapter 24). They are unique among proteins in that they are made in billions of different forms, each with a different binding site that recognizes a specific target molecule (or antigen). The precise antigen specificity of antibodies makes them powerful tools for the cell biologist. When chemically coupled to fluorescent dyes, antibodies are invaluable for locating specific molecules in cells by fluorescence microscopy (Figure 9–14).

抗体是脊椎动物免疫系统为抵御感染而产生的蛋白质（见第 24 章）。它们在蛋白质中独一无二之处在于：可以有数十亿种不同形式，每一种都带有一个不同的结合位点，能识别某一特定的靶分子（即抗原）。抗体这种精确的抗原特异性，使其成为细胞生物学家的有力工具。当与荧光染料化学偶联后，抗体对于用荧光显微镜定位细胞内的特定分子极为宝贵（图 9–14）。

There are many other membrane-enclosed organelles in eukaryotic cells. Unlike the nucleus, most of them are enclosed by single membranes. The most extensive organelle is the endoplasmic reticulum (ER), which is where most cell membrane components are made, along with materials destined for secretion to the outside of the cell. The Golgi apparatus receives these molecules from the ER and modifies and packages them for secretion or transport to another cell compartment. Lysosomes are small irregularly shaped organelles in which intracellular digestion occurs. Peroxisomes are small vesicles where hydrogen peroxide is used to inactivate toxic molecules.

真核细胞中还有许多其他膜包裹的细胞器。与细胞核不同，它们大多数由单层膜包裹。最庞大的细胞器是内质网（ER），大多数细胞膜组分以及待分泌到细胞外的物质都在这里合成。高尔基体从内质网接收这些分子，对其修饰并包装，以便分泌或转运到另一个细胞区隔。溶酶体是形状不规则的小型细胞器，细胞内消化就在其中进行。过氧化物酶体则是小囊泡，其中利用过氧化氢使有毒分子失活。

In these cells, the limited proliferation capacity reflects a progressive shortening and uncapping of the cell’s telomeres, the repetitive DNA sequences and associated proteins that cap the ends of each chromosome (discussed in Chapter 5). Human somatic cells in the body have turned off production of the enzyme, called telomerase, that normally maintains the telomeres, which is why their telomeres shorten with each cell division.

在这些细胞中，增殖能力受限反映的是细胞端粒的逐渐缩短与“失帽”；端粒即封盖每条染色体末端的重复 DNA 序列及与之相关的蛋白质（见第 5 章）。人体内的体细胞已经关闭了端粒酶——这种正常维持端粒的酶——的产生，这就是它们的端粒会随每次细胞分裂而缩短的原因。

Human fibroblasts can often be coaxed to proliferate indefinitely by providing them with the gene that encodes the catalytic subunit of telomerase; in this case, they can be propagated as an immortalized cell line. Some human cells, however, cannot be immortalized by this trick. Although their telomeres remain long, they still stop dividing after a limited number of divisions because culture conditions cause excessive stimulation of cell proliferation, which activates a poorly understood protective mechanism that stops cell division—a process sometimes called culture shock.

如果给人类成纤维细胞导入编码端粒酶催化亚基的基因，它们往往就能被诱导着无限增殖，此时可作为永生化细胞系传代。不过，有些人类细胞并不能用这一招永生化：尽管它们的端粒仍然很长，却依旧在有限次数的分裂后停止分裂，因为培养条件会过度刺激细胞增殖，从而启动一种尚未弄清的保护性机制来停止细胞分裂——这一过程有时被称为“培养休克”。

The cell cultures obtained by disrupting tissues tend to suffer from a problem—eventually the cells die. Most vertebrate cells stop dividing after a finite number of cell divisions in culture, a process called replicative cell senescence (discussed in Chapter 17). Normal human fibroblasts, for example, typically divide only 25–40 times in culture before they stop. In these cells, the limited proliferation capacity reflects a progressive shortening and uncapping of the cell’s telomeres, the repetitive DNA sequences and associated proteins that cap the ends of each chromosome (discussed in Chapter 5).

通过打散组织而获得的细胞培养物往往面临一个问题——细胞最终会死亡。大多数脊椎动物细胞在培养中经过有限次数的细胞分裂后就停止分裂，这一过程称为复制性细胞衰老（见第 17 章）。例如，正常人成纤维细胞在培养中通常只分裂 25～40 次就停止。在这些细胞中，增殖能力受限反映的是细胞端粒的逐渐缩短与去帽；端粒即位于每条染色体末端、起帽子作用的重复 DNA 序列及其结合蛋白（见第 5 章）。

Unlike yeast, most tissue cells are not adapted to living suspended in fluid and require a solid surface on which to grow and divide. For cell cultures, this support is usually provided by the surface of a plastic culture dish. Cells vary in their requirements, however, and many do not proliferate or differentiate unless the culture dish is coated with materials that cells adhere to, such as polylysine or extracellular matrix components.

与酵母不同，大多数组织细胞并不适应悬浮于液体中生存，而需要一个固体表面供其生长和分裂。在细胞培养中，这种支撑通常由塑料培养皿的表面提供。不同细胞的要求各异，许多细胞除非培养皿被包被上细胞可黏附的材料（如多赖氨或细胞外基质成分），否则不会增殖或分化。

Embryonic stem cells are an important cell type isolated from the early mammalian embryo. As described in Chapter 22, these cells are pluripotent; that is, they have the potential to differentiate into any cell type in the body. When cultured in the presence of the appropriate extracellular signaling factors and nutrients, stem cells can be directed to differentiate into a wide range of specific cell types. Under some conditions, it is even possible to stimulate these cells to assemble into three-dimensional multicellular structures that are miniature versions of certain organs, such as the gut. These organoids provide a powerful tool for the analysis of tissue function (see Chapter 22).

胚胎干细胞是从哺乳动物早期胚胎中分离出的一类重要细胞。如第 22 章所述，这些细胞是多能的，即具有分化为体内任何细胞类型的潜能。在含有适当的细胞外信号因子和营养成分的条件下培养时，干细胞可被定向诱导分化为多种特定细胞类型。在某些条件下，甚至可以刺激这些细胞装配成三维的多细胞结构，即某些器官（如肠）的微型版本。这些类器官为分析组织功能提供了强有力的工具。

Cryo-electron microscopy (cryoEM) depends crucially on very rapidly freezing the aqueous specimen to form vitreous ice, which does not allow ice crystals to form and therefore does not damage the specimen. A very thin (about 100 nm) film of an aqueous suspension of purified macromolecular complex is prepared on a microscope grid and is then rapidly frozen by being plunged into a coolant.

冷冻电镜（cryoEM）至关重要地依赖于将含水样品极快速冻结以形成玻璃态冰；玻璃态冰不允许冰晶形成，因而不会损伤样品。先将纯化的大分子复合物水悬液制成非常薄（约 100 nm）的膜，附于镜用载网上，再把它投入冷却剂中快速冻结。

When centrifuged, the various components in the mixture move as a series of distinct bands through the solution, each at a different rate, in a process called velocity sedimentation (Figure 8-6A). For the procedure to work effectively, the bands must be protected from convective mixing, which would normally occur whenever a denser solution (for example, one containing organelles) finds itself on top of a lighter one (the salt solution). This is achieved by augmenting the solution in the tube with a shallow gradient of sucrose prepared by a special mixing device.

离心时，混合物中的各种组分以不同速率向下运动，形成一系列彼此分开的条带，这一过程称为速度沉降。为使该方法有效，必须保护条带不受对流混合的干扰——只要较重的溶液（例如含细胞器的那一层）处于较轻溶液（盐溶液）之上，就会发生对流混合。解决办法是用专门的混合装置在离心管内制备一个浅蔗糖梯度。

The ultracentrifuge is also used to separate cell components on the basis of their buoyant density, independently of their size and shape. In this case, the sample is sedimented through a steep density gradient that contains a very high concentration of sucrose or cesium chloride. Each cell component begins to move down the gradient as in Figure 8–6A, but it eventually reaches a position where the density of the solution is equal to its own density.

超速离心机还可以根据细胞组分的浮力密度、而与其大小和形状无关地将它们分开。这时，样品被离心通过一个含有很高浓度蔗糖或氯化铯的陡密度梯度。每一种细胞组分都像图 8–6A 那样开始沿梯度下行，但最终会到达一个位置，在那里溶液的密度恰好等于它自身的密度。

Molecules in all kinds of thin sections can be labeled to identify and localize them. We have seen earlier how antibodies can be used in conjunction with fluorescence microscopy to localize specific macromolecules. An analogous method—immunogold electron microscopy—can be used in the electron microscope. The usual procedure is to incubate a thin section first with a specific primary antibody, and then with a secondary antibody to which a colloidal gold particle has been attached. The gold particle is electron-dense and can be seen as a black dot in the electron microscope (Figure 9–43). Different antibodies can be conjugated to different-sized gold particles so multiple proteins can be localized in a single sample.

各类超薄切片中的分子都可以被标记，以便鉴定和定位。前文已介绍了如何将抗体与荧光显微术结合来定位特定大分子；类似的方法——免疫胶体金电镜术——也可用于电子显微镜。通常的做法是先用特异性的第一抗体与超薄切片孵育，再用偶联了胶体金颗粒的第二抗体孵育。金颗粒具有电子致密性，在电镜下表现为黑点。不同抗体可偶联不同大小的金颗粒，因而可在同一样品中定位多种蛋白质。

Biological tissues are composed mainly of atoms of very low atomic number (primarily carbon, oxygen, nitrogen, and hydrogen). To make them more readily visible, tissues are often impregnated (before or after sectioning) with the salts of heavy metals such as uranium, lead, and osmium. The degree of impregnation, or “staining,” with these salts will vary for different cell constituents. Lipids, for example, tend to stain darkly after osmium fixation, revealing the location of cell membranes (see, for example, Figure 12–2 or Figure 12–15).

生物组织主要由原子序数很低的原子（主要是碳、氧、氮、氢）组成。为了使它们更易于观察，常常要用铀、铅、锑等重金属的盐对组织（在切片前或切片后）进行浸泡。不同细胞成分对这些盐的浸泡程度（即“染色”）各不相同。例如，脂质在锑酸固定后往往着色很深，从而显示出细胞膜的位置。

Because the specimen is exposed to a very high vacuum in the electron microscope, living tissue is usually killed and preserved by chemical fixation. As electrons have very limited penetrating power, the fixed tissues normally have to be cut into extremely thin sections (25–100 nm thick, about 1/200 the thickness of a single cell) before they are viewed. This is achieved by dehydrating the specimen, permeating it with a monomeric resin that polymerizes to form a solid block of plastic, then cutting the block with a fine glass or diamond knife on a special microtome. The resulting ultrathin sections, free of water and other volatile solvents, are supported on a small metal grid for viewing in the microscope (Figure 9–41).

由于样品在电镜中暴露于极高真空，活组织通常要用化学固定杀死并保存。因为电子的穿透能力很有限，固定后的组织通常必须先切成极薄的切片（25–100 nm 厚，约为单个细胞厚度的 1/200）才能观察。具体做法是：将样品脱水，用单体树脂渗透并使其聚合成固体塑料块，再用精细的玻璃刀或钻石刀在专用切片机上切开塑料块。所得的超薄切片不含水及其他易挥发溶剂，被承载在小小的金属载网上以供镜下观察。

However, we often want to examine thin sections, and the frozen tissue can be sectioned directly in a cooled microtome. A compromise is to rapidly freeze the tissue, replace the water with organic solvents, embed the tissue in plastic resin, and finally cut sections. This approach, called freeze substitution, stabilizes and preserves the tissue in a condition very close to its original living state (Figure 9–42).

不过，我们往往仍希望观察薄切片，而冻结的组织可以在冷却的切片机上直接切片。一种折中的做法是：先将组织快速冻结，再用有机溶剂置换其中的水，然后用塑料树脂包埋，最后切片。这种方法称为冷冻置换（freeze substitution），它能使组织稳定并保存在与其原来活体状态极为接近的状况中。

It is possible to fuse one cell with another to form a heterokaryon, a combined cell with two separate nuclei. Typically, a suspension of cells is treated with certain inactivated viruses or with polyethylene glycol, each of which alters the plasma membranes of cells in a way that induces them to fuse.

可以将一个细胞与另一个细胞融合，形成异核细胞（heterokaryon）——一个含有两个独立细胞核的融合细胞。典型做法是用某些灭活病毒或聚乙二醇处理细胞悬液，二者都能改变细胞的质膜，从而诱导细胞相互融合。

After a small region of the plasma membrane is selectively photobleached, the remaining fluorescent molecules diffuse rapidly within the plane of the membrane and populate the bleached region. This recovery can be followed as a function of time.

在质膜的一个小区域被选择性地光漂白之后，剩余的荧光分子会在膜平面内快速扩散，并填充到漂白区域中。这一恢复过程可以作为时间的函数加以追踪。

This treatment separates cell components by size and density: in general, the largest objects experience the largest centrifugal force and move the most rapidly. At relatively low speed, large components such as nuclei sediment to form a pellet at the bottom of the centrifuge tube; at slightly higher speed, a pellet of mitochondria is deposited; and at even higher speeds and with longer periods of centrifugation, first vesicles and then ribosomes can be collected (Figure 8–5). All of these fractions are impure, but many of the contaminants can be removed by resuspending the pellet and repeating the centrifugation procedure several times.

这一处理按大小和密度分离细胞组分：一般而言，最大的颗粒受到的离心力最大，也运动得最快。在相对低的转速下，细胞核这类大组分沉降到离心管底形成沉淀；转速稍高时，得到线粒体的沉淀；转速更高、离心时间更长时，则依次可收集到囊泡和核糖体。所有这些组分都不纯，但可以把沉淀重新悬浮并反复离心多次，以除去其中许多杂质。

The antibodies employed in microscopy are commonly either purified from antiserum so as to remove all nonspecific antibodies or they are specific monoclonal antibodies that only recognize the target molecule. When we use antibodies as probes to detect and assay specific molecules in cells, we frequently use methods to amplify the fluorescent signal they produce.

显微术中所用的抗体，通常要么是从抗血清中纯化以去除所有非特异性抗体，要么是只识别靶分子的特异性单克隆抗体。当我们用抗体作为探针来检测和测定细胞中的特定分子时，常常使用各种方法来放大它们产生的荧光信号。

A versatile and widely used technique is to couple fluorescent dyes to antibody molecules, which then serve as highly specific and versatile staining reagents that bind selectively to the particular macromolecules they recognize in cells or in the extracellular matrix. Two fluorescent dyes that have been commonly used for this purpose are fluorescein, which emits an intense green fluorescence when excited with blue light, and rhodamine, which emits a deep red fluorescence when excited with green-yellow light (Figure 9–12).

一种多用途且广泛使用的技术，是将荧光染料偶联到抗体分子上，使其成为高度特异、用途广泛的染色试剂，选择性地结合它们在细胞中或细胞外基质中所识别的特定大分子。为此常用的两种荧光染料是荧光素（受蓝光激发时发出强烈的绿色荧光）和罗丹明（受黄绿光激发时发出深红色荧光）。

The lost information in the third dimension can be recovered if we have views of the same specimen but from many different directions. The computational methods for this technique are widely used in medical CT scans. In a CT scan, the imaging equipment is moved around the patient to generate the different views. In electron microscope (EM) tomography, the specimen holder is tilted in the microscope, which achieves the same result. The specimen is usually tilted to a maximum of 60° in every direction, and in this way we can arrive at a three-dimensional reconstruction, in a chosen standard orientation, by combining different views of a single object.

如果能从许多不同方向获得同一样品的图像，丢失的第三维信息就可以被恢复。这种技术所用的计算方法已广泛用于医学 CT 扫描。在 CT 扫描中，成像设备绕患者移动以获得不同角度的图像；而在电镜断层成像（EM tomography）中，则是在镜内倾斜样品杆来达到同样效果。样品通常在各个方向上最大倾斜 60°，这样就能把同一物体的不同视图组合起来，得到一个处于选定标准取向的三维重构。

Thick plastic sections of embedded material have been used to create three-dimensional reconstructions, or tomograms (Movie 9.2), of cells, but increasingly microscopists are applying EM tomography to unstained, frozen, hydrated sections, and even to rapidly frozen whole cells or organelles.

人们曾用包埋材料的厚塑料切片来制作细胞的三维重构（即断层图，tomograms），但显微镜学家正越来越多地将电镜断层成像应用于未经染色的、冻结的、保持水化状态的切片，甚至应用于快速冻结的整个细胞或细胞器。

When small amounts of protein are present and more sensitive methods are required, gels can be treated with a silver stain, which will detect as little as 10 ng of protein in a band. For some purposes, specific proteins can also be labeled with a radioactive isotope tag; exposure of the gel to film or a radiation detector results in an autoradiograph on which the labeled proteins are visible (see Figure 8–16).

当蛋白质量很少、需要更灵敏的方法时，可对凝胶做银染色，它能检出条带中低至 10 ng 的蛋白质。出于某些目的，还可以用放射性同位素标签标记特定蛋白质；将凝胶曝光于胶片或辐射探测器，就得到一张放射自显影图，其上可见被标记的蛋白质。

The limiting separation at which two objects appear distinct—the so-called limit of resolution—depends on both the wavelength of the light and the numerical aperture of the lens system used. The numerical aperture affects the light-gathering ability of the lens and is related both to the angle of the cone of light that can enter it and to the refractive index of the medium the lens is operating in; the wider the microscope opens its eye, so to speak, the more sharply it can see (Figure 9–5).

使两个物体仍能被区分开的最小间距——即所谓的分辨率极限——取决于光的波长和所用透镜系统的数值孔径。数值孔径影响透镜的集光能力，它既与能够进入透镜的光锥角度有关，也与透镜所处介质的折射率有关；打个比方，显微镜“睁眼”睁得越大，它就看得越清楚。

The GFP DNA-coding sequence can also be inserted at the beginning or end of the coding sequence for another protein, yielding a chimeric product consisting of that protein with a new GFP domain attached. In many cases, this GFP fusion protein behaves in the same way as the original protein, directly revealing its location and activities by means of its genetically encoded fluorescence (Figure 9–18).

GFP 的 DNA 编码序列也可以插入另一种蛋白质编码序列的起始或末端，产生一种嵌合产物，即该蛋白质上附加了一个新的 GFP 结构域。在很多情况下，这种 GFP 融合蛋白的行为与原来的蛋白质相同，因而能借由其基因编码的荧光直接显示该蛋白质的定位与活动。

A major goal for cell biologists is the reconstitution of every biological process in a purified cell-free system. Only in this way can we define all of the components needed for the process and control their concentrations, which is required to work out their precise mechanism of action.

细胞生物学家的一个重要目标，是在纯化的无细胞体系中重建每一项生物学过程。只有这样，我们才能确定该过程所需的全部组分并控制它们的浓度，而这正是弄清其精确作用机制所必需的。

Using the recombinant DNA methods discussed later in this chapter, any gene can be modified to produce its protein with an extra amino acid sequence that provides a specific recognition tag, so as to make subsequent purification of the protein simple and rapid. Often the recognition tag is an antigenic determinant, or epitope, which can be recognized by a highly specific monoclonal antibody. The antibody can then be used to purify the protein by affinity chromatography or immunoprecipitation (Figure 8–11).

利用本章后面讨论的重组 DNA 方法，任何基因都可以被改造，使其产物携带一段额外的、提供特异识别标签的氨基酸序列，从而使后续的蛋白纯化变得简单而快速。这种识别标签往往是一个抗原决定簇（表位），可被高度特异的单克隆抗体所识别。随后就可以用该抗体通过亲和层析或免疫沉淀来纯化这个蛋白质。

A major disadvantage, however, of all fluorescence imaging techniques is that it is only the tagged molecules that are imaged—their cellular context remains invisible. When fluorescence imaging is combined, however, with looking at the same specimen in the electron microscope, this correlative light microscopy and electron microscopy technique, or CLEM, can allow specific target molecules to be examined in their full cellular context.

然而，所有荧光成像技术都有一个重大缺点：能成像的只有被标记的分子，它们所处的细胞环境仍然不可见。但如果把荧光成像与对同一样品的电镜观察结合起来，这种光镜-电镜相关成像技术（CLEM）就能使特定靶分子在其完整的细胞环境中得到考察。

By coupling one antibody to fluorescein and another to rhodamine, the distributions of different molecules can be compared in the same cell; the two molecules are visualized separately in the microscope by switching back and forth between two sets of filters, each specific for one dye. As shown in Figure 9–13, multiple fluorescent dyes can be used in the same way to clearly distinguish several different types of molecules in the same cell.

把一种抗体偶联荧光素、另一种抗体偶联罗丹明，就可以在同一个细胞中比较不同分子的分布；只需在两组分别适配一种染料的滤光片之间来回切换，便可在显微镜下分别观察这两种分子。如图 9–13 所示，同样可以使用多种荧光染料，以清晰地区分同一细胞中几种不同类型的分子。

The fluorescent dyes used for staining cells are visualized with a fluorescence microscope. This microscope is similar to an ordinary upright or inverted light microscope except that the illuminating light, from a very powerful source, is passed through two sets of filters—one to filter the light before it reaches the specimen, and one to filter the light obtained from the specimen. The first filter passes only the wavelengths that excite the particular fluorescent dye, while the second filter blocks out this light and passes only those wavelengths emitted when the dye fluoresces (Figure 9–10C).

用于细胞染色的荧光染料要用荧光显微镜来观察。这种显微镜与普通的正置或倒置光学显微镜相似，差别在于：来自强光源的照明光要依次通过两套滤光片——一套在光到达样品之前过滤，另一套过滤从样品得到的光。第一块滤光片只让能激发该荧光染料的波长通过；第二块则挡掉这些光，只让染料发荧时发出的波长通过。

Another approach is to use flow cytometry, which works by streaming a dilute suspension of cells past an illuminator and measuring the fluorescence of individual cells as they flow past the detector. Fluorescence values can be used to build histograms that reveal the variability in a process across a population of cells, with a broad histogram indicating higher variability.

另一种方法是使用流式细胞术：它的工作方式是让稀释的细胞悬液流过一个光源，并在细胞逐个流过探测器时测定单个细胞的荧光。所得的荧光值可用于绘制直方图，从而揭示某一过程在细胞群体中的变异情况，直方图越宽说明变异越大。

Measurements of the fluorescence intensity in the bleached area as a function of time can be plotted as a fluorescence recovery curve. From such graphs quantitative data can be derived about the rate of recovery and the fraction of fluorescent protein molecules that are either mobile or immobile.

将漂白区域内的荧光强度作为时间的函数测定后，可以绘成一条荧光恢复曲线。从这类图谱中，可以得到关于恢复速率、以及荧光蛋白分子中可动部分与不可动部分所占比例的定量数据。

This cultured mammalian cell is expressing an integral membrane protein called CD86, which is fused with a fluorescent protein. CD86 is a co-stimulatory protein present in the plasma membrane of antigen-presenting cells and is required for the activation of T cells (see Figure 24–34).

这个培养的哺乳动物细胞表达着一种名为 CD86 的质膜整合蛋白，它与一种荧光蛋白融合在一起。CD86 是一种共刺激蛋白，存在于抗原提递细胞的质膜上，是 T 细胞激活所必需的。

This detection method is very sensitive because many molecules of the secondary antibody recognize each primary antibody. The secondary antibody is covalently coupled to a marker molecule that makes it readily detectable. Commonly used marker molecules include fluorescent dyes (for fluorescence microscopy) and colloidal gold spheres (for electron microscopy).

这种检测方法非常灵敏，因为每一个第一抗体都可被许多个第二抗体分子识别。第二抗体与一种使其易于被检测的标记分子共价偶联。常用的标记分子包括荧光染料（用于荧光显微术）和胶体金颗粒（用于电子显微术）。

One of the simplest uses of GFP is as a reporter molecule, a fluorescent probe to monitor gene expression. A transgenic organism can be made with the GFP-coding sequence placed under the transcriptional control of the promoter belonging to a gene of interest, giving a directly visible readout of the gene’s expression pattern in the living organism (Figure 9–17). In another application, a peptide location signal can be added to the GFP to direct it to a particular cell compartment, such as the endoplasmic reticulum or a mitochondrion (see Figure 9–25B), lighting up these organelles so they can be observed in the living state.

GFP 最简单的用途之一是作为报告分子，即用于监测基因表达的荧光探针。可以制备这样一种转基因生物：把 GFP 编码序列置于目的基因启动子的转录控制之下，从而在活体中直接可见地读出该基因的表达模式。在另一种应用中，可以给 GFP 加上一段肽定位信号，把它定向到某一特定的细胞区隔，如内质网或线粒体，从而点亮这些细胞器，使其在活的状态下便能被观察。

Foremost among the fluorescent proteins used for these purposes by cell biologists is the green fluorescent protein (GFP), isolated from the jellyfish Aequorea victoria. This protein is encoded by a single gene, which can be cloned and introduced into cells of other species. The freshly translated protein is not fluorescent, but within an hour or so (less for some alleles of the gene, more for others) some of the amino acids undergo a self-catalyzed post-translational modification to generate an efficient fluorochrome, shielded within the interior of a barrel-like protein, which will now fluoresce green when illuminated appropriately with blue light (Figure 9–16).

在细胞生物学家为此类目的所用的荧光蛋白中，首屈一指的是从水母 Aequorea victoria 中分离的绿色荧光蛋白（GFP）。该蛋白由单一基因编码，这个基因可以被克隆并导入其他物种的细胞。刚翻译出的蛋白并不发荧光，但大约一小时后（因等位基因而或长或短），其中部分氨基酸会发生自催化的翻译后修饰，生成一个高效的发色团；它被屏蔽在桶状蛋白的内部，在适当的蓝光照射下就会发出绿色荧光。

Yet many cellular structures—from nuclear pores and ribosomes to nucleosomes and clathrin-coated pits—are much smaller than this and so are unresolvable by conventional light microscopy. However, several approaches are now available that bypass the limit imposed by the diffraction of light, and some can now successfully resolve objects as small as 10 nm, a remarkable, twentyfold improvement.

然而，许多细胞结构——从核孔、核糖体到核小体和网格蛋白包被小窝——都比这个极限小得多，因而无法用常规光学显微镜分辨。不过，现在已有几种途径可以绕开光的衍射所施加的限制，其中一些已能成功分辨小至 10 nm 的物体，相当于提高了令人惊叹的二十倍。