Cell Biology Practical TEM&SEM Khamis AL-Riyami

Electron microscope The electron microscope is a type of microscope that uses a beam of electrons to create an image of the specimen. It is capable of much higher magnifications and has a greater resolving power than a light microscope, allowing it to see much smaller objects in finer detail. They are large, expensive pieces of equipment, generally standing alone in a small, specially designed room and requiring trained personnel to operate them.

TRANSMISSION ELECTRON MICROSCOPE

Out Line Introduction Applications Sample Preparation Specifications of JEM-1400

Light Microscope Transmission Electron Microscope (TEM) Price Cheap Expensive Size and portability Small and Portable Large and requires Special room Type of Specimens Living or Dead Dead Samples Preparation Simple and Easy Lengthy and Complex Vacuum Not required Required Image color Natural color of sample maintained Black and White Source of illumination Light Electron Magnification Up to 2000X Up to 1,300,000X Resolution 200nm 0.2nm

Introduction Electron Microscopy is defined as a specialized field of sciences that employs the electron microscope as a tool. The transmission electron microscope (TEM) is a scientific instrument that uses electrons instead of light to study objects at very fine resolutions. They were developed in the 1930s when scientists realized that electrons can be used instead of light to "magnify" objects or specimens under study.

1. Electron Gun, producing a stream of monochromatic electrons. 2. This stream is focused to a small, thin, coherent beam by the use of condenser lenses. 3. This coherent beam will hit the sample and parts of it will transmitted. 4. This transmitted portion is focused by the objective lens into an image. 5. The image is passed down the column through the intermediate and projector lenses, being enlarged all the way. 6. The image strikes the phosphor image screen and light is generated, allowing the user to see the image.

Applications A TEM can be used in any branch of science and technology where it is desired to study the internal structure of specimens down to the atomic level. Biology and life sciences: Clinical diagnosis 2. Biological samples examinations (animals & plants) Nanoparticals & Material sciences

SAMPLES PREPARATION Extraction of tissue Fixation Dehydration Infiltration Embedding Polymerization Sectioning Staining Observation (Screening)

Extraction of Tissue Fresh Tissue Fragment thickness 1mm3

Fixation Primary Fixation with 2.5% glutaraldehyde in Sodium Cacodylate Buffer Secondary Fixation with 1% Osmium Tetroxide

Dehydration Dehydration with Alcohol (Acetone or Ethanol) 25% 75% 95% Absolute Alcohol

Infiltration Infiltration with Resin 1Alcohol : 1Resin Pure Resin

Embedding Embedding with Epoxy Resin

Polymerization Polymerization in oven under 60oC over night

SECTIONING Semithin 0.5um Ultrathin 60-90nm

Staining With heavy metals Urinayl Acetate Lead Citrate

Observation (Screening) Using JEOL JEM-1400 Electron Microscope

JEM-1400 Is a high performance, high contrast, 120kV TEM with excellent imaging and analytical capabilities in one compact. Acceleration voltage of 40 to 120kV.

JEM-1400 Specifications Resolution 0.2nm Accelerating Voltage 40-120KV Magnification x200-x1,200,000 Along with this instrument implanted the Tomography System

Tomography System Three-dimensional imaging techniques Electron tomography is similar in approach to larger scale medical imaging technologies such as CT scans and MRI. Tomography acquires a series of projected images from different perspectives as the sample is rotated incrementally about an axis perpendicular to the viewing direction. A computer then combines these images into a three-dimensional model of the sample. In tomographic analysis the entire acquisition of the tomographic series can be automated.

Scanning Electron Microscope

Outline Introduction Application of SEM Sample preparations SEM JSM-6510LA specifications

Introduction The scanning electron microscope (SEM) uses a focused beam of high-energy electrons interact with the surface of solid specimens. From electron-sample interactions reveal information about the sample including texture, chemical composition, and crystalline structure Qualitative and quantitative chemical analysis information is also obtained using an energy dispersive x-ray spectrometer (EDS) with the SEM

Principle of SEM A beam of high energy electrons generated by an electron gun Beam processed by magnetic lenses, focused at the specimen surface and systematically scanned across the surface of a specimen

Used to analyze characteristic X-ray by measuring energies of X-ray Principle of EDS Used to analyze characteristic X-ray by measuring energies of X-ray The detector is cooled by LN2 in order to reduce electric noise The advantage of EDS is that the X-ray from a wide range of elements from B to U are analyzed simultaneously. Can result wit semi-quantitative and qualitative analysis of the sample also result with X-ray mapping of the sample.

Applications The scanning electron microscope (SEM) gives the user with an advantage over the light microscope (LM) in three areas: Resolution at high magnification. The best resolution possible in LM is about 200 nm SEM has a resolution of better than 10 nm (typically 5 nm) . Depth of field . greater depth of field up to 100 times that of light microscopy Microanalysis. analysis of sample composition including information about chemical composition, crystallographic, magnetic and electrical characteristics.

Practical uses for the SEM SEM is used in most of the scientific fields and industrial field, in Forensic science, biology, chemistry, engineering, Geological science, archaeology and even in art.

Sample Preparations The specimen must meet the following requirements befor it is loaded to the stage: SEM has vacuum 10-4 Pa so the samples need to be dried The sample should be not more than 150mm in diameter The surface to observe is exposed. The specimen is firmly fixed to the specimen mount . The specimen has conductivity.

Critical point Draying Biological samples Fixation Dehydration Critical point Draying Coating Mounting observation

Fixation glutaraldehyde Osmium tetroxide

Critical Point Draying Dehydration Critical Point Draying Coating Mounting

Metals, ceramic and glass If the sample dry and conductive It dose not need preparation If the sample not conductive Need to coat with Platinum or gold thickness 20-30 nm

SEM specifications JEOL JSM-6510LA Observation of specimens up to 150mm in diameter. high resolution of 3.0nm at 30kV Magnification 5x to 300,000 JEOL (energy dispersive X-ray spectrometer (EDS) provides elemental analysis. Low Vacuum (4.0nm)(30Kv)) analyze non-conductive specimens without conductive coating. image display of the secondary electron image and a backscattered composition image allow the user to contrast and compare specific details

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