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
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
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.
电镜与光镜光路图比较
?= 150/V
Work V,104—105 V at 60,000V,? =0.05 ?
theoretically D = 0.61x 0.05 / 1x1= ~0.03 ?
Practically,resolution limit,3-4 ?,
for Cellular structure 10-15 ?
电镜与光镜的比较
电子显微镜与光学显微镜的基本区别
分辨本领 光 源 透 镜 真 空 成像原理
人眼 0.2mm 可见光
光学显微镜 200nm 可见光
( 波长 4 0 0 ～
700nm)
利用样本对光的吸收形成明暗
反差和颜色变化
100nm 紫外光
（波长约 200nm ）
玻璃透镜 不要求真空
电子显微镜 接近 0.1nm 电子束
( 波长 0, 0 1 ～
0.9nm)
电磁透镜 1.33 × 10
- 3
～
1.33 × 10
- 5
Pa
利用样品对电子的散射和透射形
成明暗反差
* Specimen preparation for TEM
Standard method:
Fixation,Glutaraldehyde ( proteins cross-linking),osmium
tetroxide (lipid),Dehydration (in alcohol) and
embedding within epoxy plastic called Epon
Section,0.02-0.1μ m,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)
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
,
Fr Freeze-fracture replication and Freeze etching,
rapidly freeze specimen at –150oC without fixative,
Freeze-fracture replication ( shadow a casting heavy-
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
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 ）
冰冻断裂与蚀刻复型：主要用来观察膜断裂面
的蛋白质颗粒和膜表面结构 。
快速冷冻 深度蚀刻技术 （ quick freeze deep etching）
?电镜三维重构技术
电子显微术, 电子衍射与计算机图象处理相结合
而形成的具有重要应用前景的一门新技术 。
电镜三维重构技术与 X-射线晶体衍射技术及核磁
共振分析技术相结合, 是当前结构生物学 。
（ Structural Biology） ——主要研究生物大分子空间结构及其
相互关系的主要实验手段 。
?免疫电镜技术,
?免疫铁蛋白技术
?免疫酶标技术
?免疫胶体金技术
应用：通过对分泌蛋白的定位，可以确定某种蛋白
的分泌动态；胞内酶的研究；膜蛋白的定位与骨架
蛋白的定位等
E
2) Scanning Electron microscopy (SEMs)
The means to examine in great clarity and detail the
surfaces of objects ranging in size from a virus to an
animal head keeping surface properties as the living state,
It is very important to display the outer cell surface and
various processes,extensions and extracellular materials
that are involved in the interaction of cell—its
environment.
P504
Specimen preparation for SEM.
A key procedure,critical-point drying using liquid CO2 —
dehydrate.
Dehydrated specimen is coated a layer of carbon and then metal
( gold or gold-palladium)
Image formation,in the SEM,the image is formated by the
electrons that are reflected back from the specimen (back-scattered)
or by secondary electrons given off by the specimen after being
struck by the primaty electron beam scanning the specimen,These
electrons strike a detector near the surface of specimen and are
scanning in the cathode-ray tube (as a TV screen),The result is an
image that reflects the surface topology of the specimen,because it
is this topology ( the crevices,hills and pits) that determines the
number of electrons collected from the various parts of the surface.
The most remarkable properties of the SEM,great range of
magnification,tremendous depth of focus (~500x deeper than that
of Light Microscopy) resulting their 3D image,The resolution is ~6
nm.
Other application or development of EM:
X ray micro-zone analysis
Scanning transmission electron microscope
High-volatge EM 3x106V
Scanning tunnel EM(STM),Atomic force EM etc.
三,扫描遂道显微镜
?原理,量子力学中的隧道效应
?装置：扫描的压电陶瓷, 逼近装置, 电子学反馈控制系统和
数 据采集, 处理, 显示系统 。
?特点,（ 1） 分辨本领高, （ 侧分辨率为 0.1～ 0.2nm，
纵分辨率可达 0.001nm） ；
（ 2） 可在真空, 大气, 液体等多种条件下工作；
（ 3） 非破坏性测量 。
?用途：纳米生物学研究领域中的重要工具
1,3,Cell sorting,cell culture and cell clone
Cell sorting centrifugation,
Flow cytometry sorting (FCM)
1,Cell culture
2,Cell culture condition,medium & serum-free
3,medium pH,CO2,etc.
4,Separation of cells from tissue
Primary culture and secondary culture
Cell strain & cell line
Stem cell culture and differentiation
5,Mass culture & clone culture (cell clones)
Cell fusion & cell hybridization
Plant cell culture --protoplast,callus
6.
CHO细胞的培养
5.The fractionation of a cell’s contents by
differential centrifugation
Remove tissue
Homogenate
Speed differential centrifugation
Density gradient centrifugation
Identify
6,Biochemical and Molecular biological technique
The isolation,purification,fractionation and structure
analysis of protein and nucleic acid.
DNA recombination and molecular hybridization
Gene analysis and gene cloning
Reverse genetic
Antisense RNA
Gene deletion
PCR (polymerase chain reaction)
Gene transfer into eukaryotic cell and mammalian
embryos,transgenic animal animals and plants gene
knockout mice
7,Analysis of cells by non-impairment detect
techniques
structural and functional analysis of cells and
molecules in living state (ESR,NMR,Confocal etc.)
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
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
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.
电镜与光镜光路图比较
?= 150/V
Work V,104—105 V at 60,000V,? =0.05 ?
theoretically D = 0.61x 0.05 / 1x1= ~0.03 ?
Practically,resolution limit,3-4 ?,
for Cellular structure 10-15 ?
电镜与光镜的比较
电子显微镜与光学显微镜的基本区别
分辨本领 光 源 透 镜 真 空 成像原理
人眼 0.2mm 可见光
光学显微镜 200nm 可见光
( 波长 4 0 0 ～
700nm)
利用样本对光的吸收形成明暗
反差和颜色变化
100nm 紫外光
（波长约 200nm ）
玻璃透镜 不要求真空
电子显微镜 接近 0.1nm 电子束
( 波长 0, 0 1 ～
0.9nm)
电磁透镜 1.33 × 10
- 3
～
1.33 × 10
- 5
Pa
利用样品对电子的散射和透射形
成明暗反差
* Specimen preparation for TEM
Standard method:
Fixation,Glutaraldehyde ( proteins cross-linking),osmium
tetroxide (lipid),Dehydration (in alcohol) and
embedding within epoxy plastic called Epon
Section,0.02-0.1μ m,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)
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
,
Fr Freeze-fracture replication and Freeze etching,
rapidly freeze specimen at –150oC without fixative,
Freeze-fracture replication ( shadow a casting heavy-
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
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 ）
冰冻断裂与蚀刻复型：主要用来观察膜断裂面
的蛋白质颗粒和膜表面结构 。
快速冷冻 深度蚀刻技术 （ quick freeze deep etching）
?电镜三维重构技术
电子显微术, 电子衍射与计算机图象处理相结合
而形成的具有重要应用前景的一门新技术 。
电镜三维重构技术与 X-射线晶体衍射技术及核磁
共振分析技术相结合, 是当前结构生物学 。
（ Structural Biology） ——主要研究生物大分子空间结构及其
相互关系的主要实验手段 。
?免疫电镜技术,
?免疫铁蛋白技术
?免疫酶标技术
?免疫胶体金技术
应用：通过对分泌蛋白的定位，可以确定某种蛋白
的分泌动态；胞内酶的研究；膜蛋白的定位与骨架
蛋白的定位等
E
2) Scanning Electron microscopy (SEMs)
The means to examine in great clarity and detail the
surfaces of objects ranging in size from a virus to an
animal head keeping surface properties as the living state,
It is very important to display the outer cell surface and
various processes,extensions and extracellular materials
that are involved in the interaction of cell—its
environment.
P504
Specimen preparation for SEM.
A key procedure,critical-point drying using liquid CO2 —
dehydrate.
Dehydrated specimen is coated a layer of carbon and then metal
( gold or gold-palladium)
Image formation,in the SEM,the image is formated by the
electrons that are reflected back from the specimen (back-scattered)
or by secondary electrons given off by the specimen after being
struck by the primaty electron beam scanning the specimen,These
electrons strike a detector near the surface of specimen and are
scanning in the cathode-ray tube (as a TV screen),The result is an
image that reflects the surface topology of the specimen,because it
is this topology ( the crevices,hills and pits) that determines the
number of electrons collected from the various parts of the surface.
The most remarkable properties of the SEM,great range of
magnification,tremendous depth of focus (~500x deeper than that
of Light Microscopy) resulting their 3D image,The resolution is ~6
nm.
Other application or development of EM:
X ray micro-zone analysis
Scanning transmission electron microscope
High-volatge EM 3x106V
Scanning tunnel EM(STM),Atomic force EM etc.
三,扫描遂道显微镜
?原理,量子力学中的隧道效应
?装置：扫描的压电陶瓷, 逼近装置, 电子学反馈控制系统和
数 据采集, 处理, 显示系统 。
?特点,（ 1） 分辨本领高, （ 侧分辨率为 0.1～ 0.2nm，
纵分辨率可达 0.001nm） ；
（ 2） 可在真空, 大气, 液体等多种条件下工作；
（ 3） 非破坏性测量 。
?用途：纳米生物学研究领域中的重要工具
1,3,Cell sorting,cell culture and cell clone
Cell sorting centrifugation,
Flow cytometry sorting (FCM)
1,Cell culture
2,Cell culture condition,medium & serum-free
3,medium pH,CO2,etc.
4,Separation of cells from tissue
Primary culture and secondary culture
Cell strain & cell line
Stem cell culture and differentiation
5,Mass culture & clone culture (cell clones)
Cell fusion & cell hybridization
Plant cell culture --protoplast,callus
6.
CHO细胞的培养
5.The fractionation of a cell’s contents by
differential centrifugation
Remove tissue
Homogenate
Speed differential centrifugation
Density gradient centrifugation
Identify
6,Biochemical and Molecular biological technique
The isolation,purification,fractionation and structure
analysis of protein and nucleic acid.
DNA recombination and molecular hybridization
Gene analysis and gene cloning
Reverse genetic
Antisense RNA
Gene deletion
PCR (polymerase chain reaction)
Gene transfer into eukaryotic cell and mammalian
embryos,transgenic animal animals and plants gene
knockout mice
7,Analysis of cells by non-impairment detect
techniques
structural and functional analysis of cells and
molecules in living state (ESR,NMR,Confocal etc.)
