Electron microscopy of cells: a new beginning for a new century.
Electron microscopy of cells: a new beginning for a new century.
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DOI：
10.1083/jcb.153.6.f25
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发表时间：
2001-06-11
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影响因子：
--
通讯作者：
McIntosh JR
中科院分区：
文献类型：
--
作者：
McIntosh JR
人们普遍认为，大分子的EM正在进入一个新的生产力阶段（综述见诺加莱斯和Grigorieff，2001）。但不太为人所知的是，类似的创新正在细胞EM中进行。在细胞生物学的早期，生物学的“超微结构”研究是至关重要的，但最近它们的重要性已显着减弱。这种变化主要是因为EM的经典技术所能看到的东西早已被描述;它也源于EM在时间和金钱上的成本，以及最近革命性地改变了光学显微镜的创新，使其在活细胞的研究中变得更加强大。然而，最近在EM的方法和仪器的改进，现在允许的细胞器和细胞子系统的结构，其特征在于前所未有的细节和可靠性。由于层析成像，细胞的三维（3-D）结构现在可以以5-8-nm的分辨率可视化（Frank，1995; Baumeister等人，1999年）。由于高压冷冻，即使不添加化学冷冻保护剂，也可以很好地冷冻相当大尺寸的细胞标本（Shimoni和Muller，1998）。利用这种和其他快速冷冻方法，样品在几毫秒内凝固（Gilkey和Staehelin，1986），于是它们被嵌入玻璃状冰中（Sartori等人，1993年）。细胞环境仍然是水性的，但快速冷冻已经固定了细胞的所有成分，然后才可能发生显著的重排。在这些条件下，生物结构被困在一个基本上天然的状态，冰晶，这将使生理组织变形，几乎没有时间生长。更快速冷冻的标本保留了令人印象深刻的细胞内细节（Heuser和Reese，1981）。冷冻-水合的细胞可以通过使用低温样品保持器直接在EM中检查，或者它们可以随后通过“冷冻置换”固定，在此期间，在将其包埋在适合于切片的基质中之前，在80 ℃至90 ℃下用含有化学固定剂的有机溶剂替换细胞水以稳定生物结构。冷冻替代的样品看起来类似于通过常规方法制备的EM材料，但它们更有可能在其天然状态下显示结构（参见Steinbrecht和Muller，1987; McDonald和Morphew，1993）。本文将介绍最近的进展，在3-D成像的冷冻水合细胞和那些已保存的冷冻置换。然后，我们将评估所得数据对我们理解细胞机制的影响。
It is widely acknowledged that EM of macromolecules is entering a new phase of productivity (for review see Nogales and Grigorieff, 2001). It is less well known that comparable innovations are afoot in cellular EM. Studies of biological “ultra-structure” were of key importance in the early years of cell biology, but more recently their importance has waned significantly. This change has been largely because much of what could be seen by classical techniques for EM has long since been described; it has also derived from the cost of EM in both time and money and the innovations that have recently revolutionized light microscopy, making it ever more powerful for the study of living cells. However, recent improvements in both methods and instrumentation for EM are now allowing the structure of organelles and cellular subsystems to be characterized with unprecedented detail and reliability. Thanks to tomography, the three-dimensional (3-D) 1 structure of cells can now be visualized with 5–8-nm resolution (Frank, 1995; Baumeister et al., 1999). Thanks to high pressure freezing, cellular specimens of considerable size can now be well frozen, even without the addition of chemical cryoprotectants (Shimoni and Muller, 1998). With this and other methods for rapid freezing, samples become solidified within milliseconds (Gilkey and Staehelin, 1986), whereupon they are embedded in glass-like ice (Sartori et al., 1993). The cellular milieu is still aqueous, but rapid freezing has immobilized all the cell’s constituents before significant rearrangement is possible. Under these conditions, biological structure is trapped in an essentially native state, and ice crystals, which would deform the physiological organization, have had little time to grow. More rapidly frozen specimens retain impressive preservation of intracellular detail (Heuser and Reese, 1981). Frozen-hydrated cells can be examined directly in the EM by using a low temperature specimen holder, or they can subsequently be fixed by “freeze-substitution,” during which cellular water is replaced at 80 C to 90 C by an organic solvent that contains chemical fixatives to stabilize the biological structure before it is embedded in a matrix suitable for microtomy. Freeze-substituted samples appear similar to material prepared for EM by conventional methods, but they have a greater likelihood of displaying structures in their native state (for review see Steinbrecht and Muller, 1987; McDonald and Morphew, 1993). This review will describe recent progress in the 3-D imaging of both frozen-hydrated cells and those that have been preserved by freeze-substitution. We will then evaluate the impact of the resulting data on our understanding of cellular mechanisms.
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影响因子：
64.8
作者：
Bottcher, B;Wynne, SA;Crowther, RA
通讯作者：
Crowther, RA
影响因子：
7.8
作者：
Heuser, J E;Reese, T S
通讯作者：
Reese, T S
影响因子：
7.8
作者：
LADINSKY, MS;KREMER, JR;HOWELL, KE
通讯作者：
HOWELL, KE
影响因子：
3.4
作者：
Grimm, R;Barmann, M;Baumeister, W
通讯作者：
Baumeister, W
影响因子：
2.2
作者：
LUTHER, PK;LAWRENCE, MC;CROWTHER, RA
通讯作者：
CROWTHER, RA