Imaging      A number of controls are used during normal operation:-

(1) Focus (objective lens) controls, enable the beam to be adjusted to place the first image in the plain of the diffraction lens "in focus".  This action should be carried out at double the photographic magnification to be used.

(2) Brightness or Second Condenser adjusts the intensity of the illumination on the specimen.  Its alignment is corrected during operation with the Brightness or Illumination centering controls.  Always reduce the illumination level by turning the control clockwise from focus.  In this way the best beam coherence is achieved.

(3) Spot size, should be adjusted to attain a satisfactory image quality, in relation to the magnification, i.e. high resolution requires a spot size of <2µm normal operation 1-5µm.

(4) Objective Stigmators should be used like fine focus controls, to adjust the image sharpness, again use at double the photographic magnification.

(5) Objective Aperture is used to set the image contrast, it should be inserted and aligned in the diffraction mode.

(6) Magnification varies the strength of the imaging lenses which are automatically adjusted for minimum distortion.

(7) Illumination alignment should be corrected, to the screen centre, prior to focus at each new magnification

(8) Emission current should be adjusted to enable the illumination to be used in the over focus mode (high coherence)  at the maximum magnification to be used.  Insufficient emission will limit performance.  Emission is controlled by the bias or by moving the filament forward (higher current) or backwards (lower current).

The Basic Focus Procedure- Move to double your working magnification, and correct the focus and astigmatism.  Remember to use the stigmators as fine focus controls, the procedure - focus - stigmate  - stigmate - repeat, is ideal.  Do not try to look at the whole of the image, evaluate a very small area, looking for maximum contrast as an indication of focus.  Doubling the magnification, ensures that focus and astigmatism corrections are sufficient for the working level, allowing a margin for error.

Astigmatism In The Illumination  Distortion in the shape of the illumination as the second condenser lens is taken through focus, is due to astigmatism. This problem leads at best to a fall off in image intensity, and at worst to an unevenly illuminated photograph.

Astigmatism is in most cases caused by contamination on the components of the microscope.  Contamination, on the parts of the microscope that come into contact with the electron beam, prevents the electrons from being earthed when they strike the column components.  A charge builds up on the contaminant, the level of which depends upon the size and texture of the contaminant. When the charge reaches a sufficient magnitude to discharge to earth, it will do so, returning the area to a neutral state.  The process is then repeated.  It is this charge which causes a distortion of the electron beam known as astigmatism.

The illumination, or condenser lens astigmatism, may be visualised by bringing the second condenser lens to cross over in order to image the source.  This is followed by slight desaturation of the filament to produce a spot and halo configuration which will show the astigmatic distortion.  The spot and halo may be "focused" using the second condenser lens.  This "image" is then refocused in turn with the two condenser stigmator controls.  The process of condenser focus, and then stigmator adjustment is repeated until the source is well defined: the astigmatism is then corrected.

Astigmatism In The Image  Astigmatism is by far the most common reason for an  unsatisfactory image,  A directional distortion is  placed upon the image in a micrograph taken with this defect.  The degree of distortion depends upon the magnitude of the astigmatism present, and the level of focus.

Astigmatism in an image may be introduced by a charge in different areas on and around the specimen.  These areas are listed below, the first being the most probable cause of astigmatism under normal operating conditions, and the last  being the least likely factor.

Contamination on the objective aperture may cause astigmatism, but it is dependant upon the level of accelerating voltage in use.  The higher the accelerating voltage, the easier it is for the contaminants to be penetrated, resulting in a lower the level of astigmatism (charge).  If the objective aperture is not correctly aligned on the axis of the objective lens it may create astigmatism.

The specimen holder may be responsible for astigmatism in the image, or a pulsing of the incident electron beam: see Astigmatism in the Illumination.  Contamination situated below the specimen may charge sufficiently to create astigmatism.  In some cases the problem may be dependant upon specimen stage settings which will move the charge nearer to, or further away from the beam axis.

Within the lens pole piece, contaminated surfaces on or around fixed apertures may create astigmatism.  The tip of the stigmator element is a particular problem, or even the pole piece faces themselves.

Although the aperture holder is much further away from the beam axis than the apertures themselves, it may cause astigmatism if heavily contaminated.  Dirt on the grid itself or on the specimen surface, may charge creating astigmatism or image instability.  Here again the magnitude of the defect may vary with specimen position.

While easily observable using Fresnel fringes round a hole, judging astigmatism on a routine specimen is, to many operators, a difficult task.

Gross astigmatism places a directional effect upon the image, and this is most easily observed in areas of heavy granular staining.  Focus an area of this type at double the photographic magnification.  Aim for an image which has maximum contrast.  Only look at a small area of the image.  Adjust one stigmator to obtain "focus", or maximum contrast,  in the imaged particle.  Repeat with the other stigmator.  Recheck focus with the objective lens controls, and recheck with the two stigmators.

At high magnifications (that is in excess of 100,000X) the change in focus, and hence level of astigmatism, from one magnification to another may be critical if the diffraction lens is varied as part of the magnification change.

If the specimen image is recorded under conditions of accurate focus, but with a small amount of astigmatism, the resulting micrograph will display a tartan background pattern.  This pattern is easily confused with other imaging problems, and should be compared with specimen movement and alignment errors.

Charging  If any part of the specimen is in poor contact with the support grid, or the grid itself is in poor contact with the holder, or the holder is in poor contact with the stage, the specimen will not be well grounded, and will therefore charge.

A perfectly even charge simply makes the specimen unstable, causing the specimen to drift either when the stage is moved or, when the illumination level is changed.  If the charge builds up on a slightly damaged area of a specimen, zones on either side of the damage may repel one another, causing the specimen to break.

Any technique which reduces the beam current (the number of electrons reaching the specimen) reduces the specimen charge.  Lower the emission current, use a smaller spot size, or use a smaller condenser aperture.  These charge effects will also be reduced if the accelerating voltage is increased, as this allows a more rapid passage of the beam through the specimen.

Coherence  If the transmission electron microscope is operated under conditions where the coherence of the illuminating system is ignored, images that do not depict a clear focus will be produced.  Lack of coherence is displayed as an image softness, and this may lead to a misinterpretation of results: images lacking beam coherence look very similar to those of sections that are "too thick".

If the condenser system is operated to form a small spot size, and the second condenser is used in an over focus condition, the instrument will then provide coherent illumination.  The smaller the spot size, and the more the second condenser is over focused, the greater the beam coherence.  Small condenser apertures also add to the beam coherence.

Contamination  Any surface that is warmed by the passage of the electron beam is subject to contamination.  Residual gases within the vacuum system are cracked by the heat of the beam, forming a  hydrocarbon layer on these surfaces.  In the case of the specimen this is very critical.  The hydrocarbon forms on both upper and lower surfaces, increasing the specimen  thickness considerably.  Contamination will be recognised as a dark circle on the specimen which is visible when the magnification is lowered.  A contamination rate of 6nm per minute is not uncommon if precautions are not taken.  As specimen thickness limits resolution (Cosslett, 1956 Resolution = 1/10 thickness), contamination will limit an instruments performance on two counts, through an increase in specimen  thickness, and through contrast limitation.

There are a number of areas where the operator has an influence over the rate of contamination within an instrument.  For example the deposition of contamination within an instrument relates to the vacuum level.   The better the vacuum, the lower the level of contamination.  All microscopes should be run for 24 hours a day, for seven days a week where possible.  Any form of grease or moisture will act as a source for contamination.  All internal components of the microscope, including the specimen holder, should be handled with gloved hands and all photographic materials should be thoroughly outgassed, prior to being placed within the microscope's vacuum system.  Vacuum grease should only be used on moving seals, and then only an absolute minimum should be used.

Most manufacturers fit anti contamination devices on their microscopes, and if fitted they are intended to be used.  A cold surface will adsorb gas molecules, and if placed near to the specimen, will dramatically reduce the rate of contamination.  The cold trap should be filled with liquid nitrogen (solid carbon dioxide and alcohol, or acetone, may be substituted if nitrogen is not available) at least thirty minutes prior to operating the instrument, and kept filled throughout the operating period.  If a cold finger is allowed to warm during operation etching of the specimen may take place: see Etching.

If a small spot size is used, the area of specimen illuminated by the beam is reduced, preventing damage to areas prior to their observation.  In the same way a small condenser aperture will help to reduce contamination.  If a small objective aperture is used, this will limit the pathway of contamination migrating up the column to the specimen.  The source is the camera system which is the dirtiest part of the microscope.

Contrast  In a transmission electron microscope, the image contrast is usually a combination of amplitude and phase contrast or diffraction contrast.  In the investigation of biological thin sections, amplitude contrast is the main consideration.  As the structures (e.g. organelles) being evaluated become finer, the contribution of phase contrast is increased, being the sole source of contrast when very fine low density materials are being observed.

A number of areas within the operation of the microscope have a dramatic effect upon the image contrast.  Biological transmission electron microscopy was initially carried out at 50 or 60kV, depending upon the kV range of the instrument.  Improvements in embedding media and a greater awareness of the improvement in image quality through the use of higher accelerating voltages, have resulted in a move towards greater use of these higher voltage levels.  The higher the accelerating voltage, the higher the energy of the electron beam, and the easier it is able to pass through the specimen, thereby reducing image contrast.  Most thin section microscopy may be carried out with adequate contrast when operating at 80kV.  It is not uncommon for work  on sections under 120nm thick to be carried out at 120 or 125kV (Heap, et al., 1983).  Artifacts may be introduced into an image through too low an accelerating voltage, and the true interpretation of a stained area may become confused because there is too much contrast.

In material science, particularly when working with metals, the tilting system plays a large part in adjusting the orientation of the specimen, to maximise the appropriate diffraction contrast.  It is therefore  to ensure that the stage is correctly set at the eucentric position and that before tiling the tilt speed is at the desired level.

The conventional design criteria for an objective lens are directed toward the highest resolution.  Unfortunately, a high resolution objective lens does not provide the highest contrast levels for thin section microscopy.  High resolution dictates a short focal length and minimum spherical aberration.  However, optimum contrast for the biologist may require a longer focal length, the resulting increase in spherical aberration aids image contrast.  An increase in objective lens focal  length also increases the image contrast through a decrease in the angular aperture of the objective (Lovell and Chapman, 1975).

The smaller the objective aperture the greater the image contrast.  Problems occur if the aperture is made smaller than 20µm.  This is due to the need for alignment accuracy, and the greater effect of aperture contamination, as the contaminants are being deposited very near to the beam axis.  Small apertures are also less stable in the electron beam leading to aperture shadow effects on the image.

In order to induce the minimum amount of damage to the  specimen, and to obtain suitable illumination levels for operation, it is necessary to use the highest accelerating voltage that still enables focus to be attained comfortably.  Visual contrast is of no importance, other than to view and focus the specimen: the screen is a means to end not the result!  Any contrast loss compared with low kV techniques, will be more than corrected through the use of optimised photographic procedures.  In general, one kV step higher than conventionally used in your laboratory is not excessive.  The instrument may take a short while to stabilise at a high kV, but its performance will be improved.

With eucentric side entry systems, the objective focal length may be increased by raising the specimen out of the objective lens, using the Z or eucentricity adjustment.  The specimen is moving out of the lens when the focus controls require to be turned anticlockwise to reach focus.  Side entry systems which do not incorporate a eucentric tilting system may not be modified in this way.  Conversely lowering the specimen further into the lens field will enable those pushing the instrument to its limit to attain higher resolution levels, as the aberrations decrease with higher strength lenses. There will also be an increase in magnification under these conditions, the objective lens working harder in order to focus the lowered specimen.

Etching  The removal of specimen material may occur through an increase in the level of residual gases within the specimen area.  The specimen is attacked by ionised gas and the  material removed into the vacuum.  The electron beam provides the energy for the reaction to occur, moisture usually being the catalyst.  Vacuum leaks in the specimen area, which will be undetected by the vacuum gauge, are the major cause of specimen etching.

Etching may also occur if the anti contamination cold finger is too close to the specimen.  When the finger is not correctly cooled, such that its temperature rises to around minus 80 degrees Centigrade material is removed from the specimen and onto the cold surface.  For this reason it is most important that the cold finger is cooled down well before operation, and that it is replenished until operation is complete.  Do not forget to remove specimens from the instrument after an operating session, or they may become damaged by this phenomenon.

Focus Setting Variations  The adjustment of the focal length of the objective lens in order to bring the specimen into focus, places the first image at the critical point of the diffraction/intermediate lens.  This is the condition of "true focus", the level where there would be no Fresnel fringes formed at any image point.  If the illuminating beam is rocked over a small angle (focus wobbler), any image movement at the centre of the field of view, or splitting of the image, indicates that the specimen is not at "true focus".  This focal position is the condition for maximum resolving power, but this may not coincide with the condition for maximum image contrast.

Fresnel fringes enhance contrast either side of true focus, with the point of maximum contrast occurring in an under focus condition.  The point of maximum contrast, often known as "optimum under focus" or o.u.f., will vary in relation to a number of specimen and instrument parameters.

1)  As the magnification is increased, the amount of defocus required to attain the optimum underfocus condition decreases.

2)  The thicker the section the greater the amount of underfocus required.

3)  A variation in the concentration of structures (e.g.  organelles) within a specimen will require a varied level of defocus, e.g. kidney will require less defocus than plant material where the organelles are well spaced.

4)  The accelerating voltage, and therefore the ease a which the beam penetrates the specimen will relate to image contrast and therefore optimum under focus.

5)  Image contrast will relate to the coherence of the illumination (condenser aperture size), and to the diffraction contrast (objective aperture size).

6)  Image contrast varies with the focal length of the objective lens, see Contrast.

(c) Steve Chapman Protrain 2002