Chapter 4

If two objects imaged by a camera are separated by an angle small enough that their Airy disks on the camera detector start overlapping, the objects can not be clearly separated any more in the image, and they start blurring together. Two objects are said to be just resolved when the maximum of the first Airy pattern falls on top of the first minimum of the second Airy pattern

Beam-Sample Interaction Volume

Volume of Signal Generation is greater than the area of intersection of the Beam with the Sample

Distribution of Secondary and Backscattered Electron Energies Emitted from Sample

The Electron Gun -emitter -Wehnelt cap or grid -anode -first lens in the SEM

Emitter/Wehnelt cap assembly [from Hitachi S2300]

Anode [Hitachi S2300]

Scanning Beam Mode Instruments Scanning Electron Microscope

-surface imaging & compositional mapping -microbeam chemistry -versatile sample size shape & [relative] ease of sample prep

Cross-section of Electromagnetic Lens from an SEM*

-note the copper wire turns & power leads
*dont try this at home

Final Lens

Backscatter Detector

Secondary Detector

SEM Sample Chamber [AMRAY 1830] showing positions of SE and BSE Detectors and the Final Lens

SEM Vacuum System

-multistage system: 1.) low-vac [roughing] 2.) high-vac [diff pump]
Oil Diffusion pump -high-vac

[Rotary] Mechanical pump -roughing or low-vac

Specimen and Electron Detector Geometries:

-position of detectors is a function of relative energies of the electrons

SEM Imaging SystemDetailed Schematic

Putting it all togethermaking the image:

Scanning Electron Micrograph of the surface of a kidney stone showing tetragonal crystals of Weddellite (calcium oxalate dihydrate) emerging from the amorphous central part of the stone. Horizontal length of the picture represents 0.5 mm of the figured original

The illuminating system consists of the electron gun and condenser lenses that give rise to and control the amount of radiation striking the specimen.

A specimen manipulation system composed of the specimen stage, specimen holders, and related hardware is necessary for orienting the thin specimen outside and inside the microscope.

Major Column Components of the TEM* Component Illumination System Electron Gun Condenser Lens 1 Condenser Lens 2 Condenser Aperture Gun, Source C1, Spot Size C2, Brightness C2 Aperture Generates electrons and provides first coherent crossover of electron beam Determines smallest illumination spot size on specimen Varies amount of illumination on specimenin combination with C1 Reduces spherical aberration, helps control amount of illumination striking specimen Synonyms Function of Components

Specimen Manipulation System Specimen Exchanger Specimen Stage Imaging System Objective Lens Objective Aperture Intermediate Lens Diffraction Lens Forms, magnifies, and focuses first image Controls contrast and spherical aberration Normally used to help magnify image from objective lens and to focus diffraction pattern Specimen Air Lock Stage Chamber and mechanism for inserting specimen holder Mechanism for moving specimen inside column of microscope

Intermediate Aperture Diffraction Aperture, Selects area to be diffracted Field Limiting Aperture Projector Lens 1 Projector Lens 2 P1 P2 Helps magnify image, possibly used in some diffraction work Same as P1

Observation and Camera Systems Viewing Chamber Binocular Microscope Camera Focusing Scope Contains viewing screen for final image Magnifies image on viewing screen for accurate focusing Contains film for recording

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Objective aperture located between upper and lower parts of polepiece, just under the specimen. The major function of the aperture is to help remove peripherally deflected electrons to enhance image contrast. In addition to the specimen and objective aperture, a chilled anticontaminator blade may also be inserted just above the specimen (or sometimes above and below the specimen) to prevent contaminants from condensing on specimen.

 TEM, has magnification and resolution capabilities that are over a thousand times beyond that offered by the light microscope. It is an instrument that is used to reveal the ultrastructure of plant and animal cells as well as viruses and may provide an image of the very macromolecules that make up these biological entities. The TEM is a complex viewing system equipped with a set of electromagnetic lenses used to control the imaging electrons in order to generate the extremely fine structural details that are usually recorded on photographic film. Since the illuminating electrons pass through the specimens, the information is said to be a transmitted image. The modern TEM can achieve magnifications of one million times with resolutions of 0.1 nm.

TEM Modes

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Darkfield
The darkfield mode can be used to enhance contrast in certain types of unstained specimens (thin frozen sections) or in negatively stained specimens.

(top) Darkfield image obtained by tilting illumination system. (bottom) Same specimen viewed in standard brightfield mode. Specimen consists of inorganic salt crystals.

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Diffraction
Since the crystalline lattice diffracts electrons to form bright spots on the viewing screen (similar to the mirrored rotating sphere sometimes used in ballrooms to reflect a light source onto the walls), the image will consist of a central, bright spot surrounded by a series of spots, which are the reflections. The central bright spot represents nondiffracted rays while the peripheral spots represent rays diffracted at various angles.

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The scale of things

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Staining with these heavy atoms is called "negative staining" because one sees not the object itself, but rather an area empty of stain surrounded by stain. This area is empty of stain because the sample material (protein for example) prevents it from depositing onto the carbon layer. Good contrast is achieved when the stain completely surrounds the specimen and neighboring 63 area.

Electron Microscopy
Preparation of Specimens
Preserved by fixation
1st, glutaraldehyde
Covalently cross-links proteins

2nd, osmium tetroxide

Binds to and stabilizes lipid bilayers and proteins

Tissue dehydrated, permeated with a polymerizing resin & sectioned into ultra-thin sections
50 100 nm thick (1/200 thickness of a cell)

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sectioning

knife of cleeved glass

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Immunogold Electron Microscopy

Used to visualize specific proteins
Incubate thin section with primary antibody
Then incubate with secondary antibody to which colloidal gold has been attached
Gold is electron dense and shows up as black dots

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Metal shadowing (platinum) and observe under TEM

Individual marcomolecules can been seen

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SAMPLE PREPARATION FOR SEM

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Tobacco Mosaic Virus

HighHigh-Resolution Transmission Electron Microscopy

HRTEM is an imaging mode of the transmission electron microscope (TEM) that allows the imaging of the crystallographic structure of a sample at an atomic scale. Because of its high resolution, it is a valuable tool to study nanoscale properties of crystalline material such as semiconductors and metals. At present, the highest resolution realized is 0.8 ngstrms (0.08 nm). At these small scales, individual atoms and crystalline defects can be imaged. Since all crystal structures are 3-dimensional, it may be necessary to combine several views of the crystal, taken from different angles, into a 3D map. This technique is called electron crystallography.

Ultra-small super paramagnetic iron oxide particles imaged by high resolution transmission electron microscopy. The particles have crystalline cores of magnetite 5nm in diameter surrounded by a shell of dextran. These particles are injected intravenously into patients with carotid artery disease. They accumulate at sites of inflammation and the disease state can be imaged non-invasively by MRI to determine if surgical intervention is needed.

Why HRTEM:
High resolution electron microscopy can resolve object details smaller than 1 nm (10-9 m). It can be used to image the interior structure of the specimen (comparing to atomic resolution scanning tunneling microscopy, only at the surface). Comparing to atomic resolution provided by X-ray diffraction (averaging information), HREM can provide information on the local structure. Direct imaging of atom arrangements, in particular the structural defects, interface, dislocations.

In the field of Transmission Electron Microscopy, phase contrast imaging may be employed to image columns of individual atoms. This ability arises from the fact that the atoms in a material diffract electrons as the electrons pass through them (the relative phases of the electrons change upon transmission through the sample), causing diffraction contrast in addition to the already present contrast in the transmitted beam. Phase-contrast imaging is the highest resolution imaging technique ever developed, and can allow for resolutions of less than one angstrom (less than 0.1 nanometres). It thus enables the direct viewing of columns of atoms in a crystalline material. The interpretation of phase-contrast images is not a straightforward task. Deconvolving the contrast seen in an HR image to determine which features are due to which atoms in the material can rarely, if ever, be done by eye. Instead, because the combination of contrasts due to multiple diffracting elements and planes and the transmitted beam is complex, computer simulations are used to determine what sort of contrast different structures may produce in a phase-contrast image. Thus, a reasonable amount of information about the sample needs to be understood before a phase contrast image can be properly interpreted, such as a conjecture as to what crystal structure the material has. Phase-contrast images are formed by removing the objective aperture entirely or by using a very large objective aperture. This ensures that not only the transmitted beam, but also the diffracted ones are allowed to contribute to the image. Instruments that are specifically designed for phase-contrast imaging are often called HRTEMs (high resolution transmission electron microscopes), and differ from analytical TEMs mainly in the design of the electron beam column. Whereas analytical TEMs employ additional detectors attached to the column for spectroscopic measurements, HRTEMs have little or no additional attachments so as to ensure a uniform electromagnetic environment all the way down the column for each beam leaving the sample (transmitted and diffracted). Because phase-contrast imaging relies on differences in phase between electrons leaving the sample, any additional phase shifts that occur between the sample and the viewing screen can make the image impossible to interpret. Thus, a very low degree of lens aberration is also a requirement for HRTEMs, and advances in spherical aberration (Cs) correction have enabled a new generation of HRTEMs to reach resolutions once thought impossible.

The Role of an Optical System

g ( r ) = f ( r ' )h ( r r ' )dr ' = f ( r ) h( r )

means convolution which Indicates the two functions are folded together

h(r) is the point spread function

 In reciprocal space

G (u ) = H (u ) F (u )
The factors contributing to H(u) include: Aperture Attenuation of the wave Aberration of the lens The aperture function A(u) The envelope function E(u) The aberration funciton B(u) B(u) = exp [-i (u)] This limits the resolution!

What is phase contrast transfer function? The exit wave now passes through the imaging system of the microscope where it undergoes further phase change and interferes as the image wave in the imaging plane.However, the recorded image is NOT a direct representation of the samples crystallographic structure. For instance, high intensity might or might not indicate the presence of an atom column in that precise location. The relationship between the exit wave and the image wave is a highly nonlinear one and is a function of the aberrations of the microscope. It is described by the contrast transfer function.