1. TEM 之構造與基本原理 (Principle of TEM) 2. TEM 之標本製備 (Specimen preparation for TEM)

Transcription

1 Transmission Electron Microscope (TEM) 穿透式 Scanning Electron Microscope (SEM) 掃瞄式 電子顯微鏡之構造與基本原理 (TEM 及 SEM) 電子顯微鏡標本之基本製作 (TEM 切片及 SEM 標本 ) 電子顯微鏡術在細胞分子生物學之應用 1. TEM 之構造與基本原理 (Principle of TEM) 2. TEM 之標本製備 (Specimen preparation for TEM) Molecular Imaging 分子影像醫學 7/26,27 10:00-12:00 Reference: 1. Bozzola J.J. and Russel L.D. (1999) Electron microscopy 2 nd ed., Jones and Bartlett Publisher, Sudbury, Massachusetts, USA 2. Sommerville J and Scheer U (1987) Electron microscopy in molecular biology, a practical approach. ILR Press, Oxford

2 Linear Equivalents 1 Angstrom 10 Angstroms = 0.1 nanometer = 1.0 nanometer (nm) [formerly millimicron (mμ)] 1000 nanometers = 1.0 micrometer (μm) [formerly micron (μ)] 1000 micrometers 1000 mililimeters = 1.0 millimeter (mm) = 1 meter (m) 1. Resolution (resolving power) 2. Numeral Aperture 3. Focal depth 4. Field depth camera 解像力 (Resolution) Resolving power

3 解像力 (Resolution) Resolving power The ocular lens of microscope does not really affect the resolution. n =1.5, sin 64 =0.9, λ=380 nm (UV), then d = 0.172μm 0.2μm Resolution of eye versus instrument Human naked eyes Birght field microscope SEM TEM Theoretical Tissue section Distance between Resovable points 0.2 mm 0.2 μm 0.2 nm 0.2 nm nm 1.0 nm

4 λ=h / mv. (1) de Broglie equation λ= wavelength of particle; h = Planck s constant; m = mass of particle; v = velocity of the particle. For electrons, kinetic energy = ½ mv 2 =ev. v = 2eV / m λ= h 2 / 2meV h= 6.6 x erg. sec m= 9.1 x g e = 4.8 x e.s.u. λ= [150/V] ½ 10 8 cm = 1.23 / V nm For EM, sin α = α when α is small (α = 0.01), n = 1 at vacuum, and 60 KV, d = nm (theoretical) practical: 0.2 nm At 150 kv, λ= nm At 60 kv, λ= nm Lens abbreviations: spherical abbreviation, chromatic abbreviation

5 Field depth and focal depth

6 穿透式電子顯微鏡之構造與基本原理

7 穿透式電子顯微鏡之構造與基本原理

8 Ultra-High voltage Electron Microscope (1 MV, 1000KV) University of Colorado, Boulder, Colorado 超高圧電子顕微鏡 JSM-ARM1300/1000 点分解能 : 0.10nm (1,300kV)/ 0.13nm (1,000kV) 加速電圧 : 200~1,300kV (1,000kV) 倍率 : 150~1,500,000

9 1000 KV 200KV 100KV

10 Basic Components of the Electron Microscope I. Main body (Column) II. 1. Illuminating system 2. Specimen chamber 3. Image forming system 4. Image recording system Vacuum System III. Electric System IV. Cooling system

11 Field Emission Gun Field Emision : Under strong electric field (10-9 V m -1 ), electrons will be extracted from a metal filament even though its temperature is below that for appreciable thermoionic emission. When emitter is cold, this type of emission is called field emission.

12 Alignment of Illuminating System Source alignment -- filament Change filaments (filament saturation) Symmetric spot (gun alignment) -- gun horizontal movement -- gun tilt Condenser alignment --

13 Rotary Pump atm, Pirani guage

14 Diffusion Pump 10-4 ~10-5 atm, Penning guage Basic Components of the Transmission Electron Microscope I. Main body (Column) 1. Illuminating system 2. Specimen chamber 3. Image forming system 4. Image recording system II. Vacuum System III. Electric System IV. Cooling system

15 I. Main I. Main body body (Column) (Column) 1. Illumination 1. Illumination system system A. Electron A. Electron gun gun B. Condenser B. Condenser lens (C1, lense C2) (C1, C2) 2. Specimen 2. Specimen chamber chamber 3. Image 3. Image forming forming system system A. Objective A. Objective lens lens B. Intermediate B. Intermediate lens (I1. lens I2) (I1. I2) C. Projector C. Projector lens (P1, lens P2, (P1, P3) P2, P3) 4. Image 4. Image recording recording system system A. Fluorescence A. Fluorescence screen, screen, B. Binocular B. Binocular microscope microscope C. Camera C. Camera system system

16 Apertures 1. Condenser aperture 2. Objective aperture Voltage Alignment (Alignment of Imaging System) Objective lens alignment Current alignment Voltage alignment** Intermediate lens alignment Trimming of the alignment Diffraction button Objective aperture alignment Projection lens Correction of lens

17 Correction of lens Astigmatism.. 1. Fringe (Fresnel diffraction fringe) 2. Under focus (better contrast) 3. In focus 4. Over focus

18 Side entry Specimen Holder

19 Lens system Imaging system Vacuum System Specimen changing system Power Supply Image recording system

20 Specimen Preparation for Electron Microscopy Fixation Buffer wash Postfixation 4% Paraformadehyde, 2.5% glutaraldehyde TEM Transition fluid Propylene oxide Phosphate, cacodylate ph Alcohol dehydration SEM 1% OsO 4 Critical point dryer (CPD) Alcohol, acetone Infiltration Embedding Epoxy resin Thin section and staining Sputter coating (Au+C) Mounting on the Stage 4% paraformaldehyde, Karnovsky fixative (2% paraform- & 2% glutar-aldehyde aldehyde)

21 Ultra-microtome

22 1. Uranyl acetate 2. Lead citrate 1μm thick plastic (epxoy resin) section, toluidine blue stain

23 Hepatic sinusoid 70nm thin section, uranyl acetate & lead citrate 電子顯微鏡之構造與基本原理 (TEM 及 SEM) 電子顯微鏡標本之基本製作 (TEM 切片及 SEM 標本 ) 電子顯微鏡術在細胞分子生物學之應用 SEM 之構造與基本原理 (Principle of SEM) SEM 之標本製備 (Specimen preparation)

24 Basic Principle & Components of Scanning Electron Microscope 1. Secondary electrons, 2. Scanning devices, 3. CRT recording devices I. Main body (Column) 1. Illuminating system 2. Specimen chamber 3. Image forming system 4. Image recording system II. Vacuum System III. Electric System IV. Cooling system 掃瞄式電子顯微鏡之構造與基本原理

25 本學科電子顯微鏡 : JSM-T330A (1989) 加速電壓 :5-30 KV 解像力 :10 nm

26 Specimen Preparation for Electron Microscopy Fixation Buffer wash Postfixation 4% Paraformadehyde, 2.5% glutaraldehyde TEM Transition fluid Propylene oxide Phosphate, cacodylate ph Alcohol dehydration SEM OsO 4 Critical point dryer (CPD) Alcohol, acetone Infiltration Embedding Epoxy resin Thin section and staining Sputter ion coating (Au+C) Mount on the stub Mounting on the Stage

27 SEM Specimen preparation The surface tension could be reduced by substitution of a liquid with a lower surface tension with thereby reduced damage during air-drying.

28 Critical Point The continuity of state for which there is no apparent difference between the liquid and gas state of a medium, the surface tension between this interface reduced to zero. This occurs at a specific temperature and pressure with resulting density, and is known as the Critical Point. 31 C and 1073p.s.i. (72.9 atm) -56 C and 75p.s.i. (5.10 atm)

29 CPD dried air dried Critical Point Dryer

30 Stubs (specimen stage) Sputter Ion Coater- Au Evaporator --C

31

32 Cryo-Electron Microscopy Cryo-sectioning, TEM Direct for immuno-electrron microscopy Cryo-Electron microscopy For determination of structure of macromolecule proteins (enzymes) 1. Cryo-EM

33 This picture shows an animal virus called ORF, imaged by cryo-em Cyro-EM (3D-EM) 1. Cryo-Electron Microscopy (cryo( cryo-em) is a technique for getting 3D structures of biological molecules from an electron microscope. 2. Single-particle cryo-em gathers many images of the same particle (molecule or molecular complex) in various orientations, and then uses something like computed tomography to reconstruct the 3D structure. 3. Cryo-EM routinely determines structures to about 15 Angstrom resolution, but with enough images (100,000 or more) resolutions of 7 or 8 Angstroms should be attainable. 4. So a key problem is automatically finding the particle images inside the micrographs.

34 Cryo-EM (3D EM electron tomography) The 3D structure of an object in its entirity is derived from a series of 2D images recorded of an objected tilted over various tilt angles. The data collection is tedious and involves tilting of the specimen in the electron microscope, correcting for lateral shift and change of defocus, followed by taking an image. This cycle is repeated, typically, over +/- 70 degrees, with 2 degree tilt increments. 5. 3D EM electron tomography

35 Cellular Compartments Nature Structural Biology 10, (2003) Reovirus polymerase 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 Å. By:Xing Zhang et al.,

36 Atomic force microscope The atomic force microscope (AFM) is one of the most powerful tools for determining the surface topography of native biomolecules at subnanometer resolution the AFM allows biomolecules to be imaged not only under physiological conditions, but also while biological processes are at work. Because of the high signal-to-noise (S/N) ratio, the detailed topological information is not restricted to crystalline specimens. Hence single biomolecules without inherent symmetry can be directly monitored in their native environment two dsdna molecules overlapping each other, imaged in contact under isopropanol single DNA strand

37 1.5μm x 1.5μm cytoplasmic side nucleolasmic side

38 SUMMARY 電子顯微鏡之構造與基本原理 (TEM 及 SEM) Resolution, components of EM, accelerating voltage, Transmitted electons (TEM), secondary electrons (SEM) Rotary pump & pirani guage (10-1 atm), diffusion pump & penning guage (10-4 atm) Tungsten filaments, LaB 6, cold field emission, 電子顯微鏡標本之基本製作 (TEM 切片及 SEM 標本 ) Thin sections (TEM) double staining, negative staining, spreading (virus particle) Critical point dryer (CO 2 ), ion coater, 電子顯微鏡術在細胞分子生物學之應用 Cryo-Electron Microscopy - 3D EM spicemen tilt, lateral shift, change focus, image, image analysis 原子力顯微鏡 AFM (atomic force microscopy)