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metal layer and then a carbon layer is deposited on the fracture
surface, then the Freeze etching ( a layer of ice can evaporate from
the surface) and rapid-freeaing, deep-etching(-269 oC)specimen is
differential scattering of electron by parts of the specimen. The later is proportional to the amount of matter present in that part, that is its mass thickness, which is a measure of the number of atoms per unit area and their atomic density.
removed)
主要电镜制样技术
超薄切片技术 用于电镜观察的样本制备示意图 负染色技术（Negative staining）
染色背景，衬托出样品的精细结构
金属投影 冰冻蚀刻技术（Freeze etching） （技术示意图）
电镜与光镜的比较
电子显微镜与光学显微镜的基本区别
人眼 光学显微镜
分辨本领
光源
透镜
0.2mm 200nm
可见光
可见光 ( 波长 400 ～ 700nm)
100nm
紫外光
玻璃透镜
（波长约200nm）
真空
成像原理
利用样本对光的吸收形成明暗 反差和颜色变化
不要求真空
电子显微镜
接近0.1nm
电子束
( 波 长 0.01 ～
.
Fr
Freeze-fracture replication and FreeБайду номын сангаасe etching:
rapidly freeze specimen at –150oC without fixative.
Freeze-fracture replication ( shadow a casting heavy-
tetroxide (lipid), Dehydration (in alcohol) and embedding within epoxy plastic called Epon Section: 0.02-0.1μm, cut by glass or diamond Section mounting on grids and stain by electron dyes, which provide the mass thickness required to scatter the electron beam: uranyl acetate and lead citrate, or be treated with metal-tagged antibody ect. Or a number of cytochemical procedures to detect some enzymes as acid phosphatase.
P505
*Special techniques:
N
Negative staining: for very small particulate materials,
which collects little stain. ( uranyl acetate, potassium
phosphotungatate)
电镜与光镜光路图比较
= 150/V
Work V: 104—105 V
at 60,000V, =0.05 Å
theoretically D = 0.61x 0.05 / 1x1= ~0.03 Å Practically, resolution limit: 3-4 Å,
for Cellular structure 10-15 Å
电子显微镜的基本构造
*EM structure:EM consist largely of
(1) A cylindrical column (a) Cathode, tungsten wire filament, a source of electron (b) Electromagnet lens, 1000-250,000 X magnification (c) Screen, photographic plate, or film (2) Vacuum system (3) Electronically control console prowerful suppler 104-105V The formation of an image in the TEM depends on the
0.9nm)
电磁透镜
1.33×10-3～ 利用样品对电子的散射和透射形 1.33×10-5Pa 成明暗反差
* Specimen preparation for TEM
Standard method: Fixation: Glutaraldehyde ( proteins cross-linking), osmium
2. Electron microscopy
Transmission Electron microscopy (TEMs) Scanning Electron microscopy (SE
1) Transmission Electron microscopy (TEMs) 1931 Ruska 1939 Siemens commercialisation, 1945 used in cells, OsO4 1948 Peale Baker bio-section
Shadow casting: to evaporate heavy metal and deposit
a metallic coat over the accessible surfaces of specimen. Heated
filament is composed of platinum together with carbon
