Glossary

Acronyms in the Tecnai manuals
LM: Low Magnification mode. In this mode the objective lens is only weakly excited (<10%). The diffraction lens (first lens below objective) is now used as the main imaging lens. The diffraction pattern of the weakly excited objective lens is now at the SA plane.

HM: High Magnification mode. In this mode the objective lens is strongly excited (>80%) and is the main imaging lens. The diffraction lens focuses at the image plane of the objective lens.

Mi, SA and Mh: Three ranges in the HM mode. In the SA range (2.6 - 390 kx on L120C) the first intermediate image is at the selected area(SA) aperture height. In Mi range and the Mh range, the first intermediate image is below and above the SA aperture, respectively (Reference: Tecnai manual - Modes 12 to 30). When in the Mi range, the TEM software displays the magnification as "M nnnn ×". In the SA range and the Mh range the magnifications are displayed as "SA nnnn ×" and "Mh nnnn ×" respectively.

D: Diffraction mode. The diffraction lens focuses at the back focal plane of the objective lens (the fixed height for the objective aperture).

DF: Dark Field. The axial direct beam is blocked by the objective aperture and only some of the scattered electrons are used for forming the image.

BF: Bright Field. Both the axial direct beam and the scattered electrons that pass the objective aperture are used for forming the image. The size of the objective aperture controls the contrast (amplitude contrast or mass-thickness contrast).

nP: nano probe mode. In this mode the mini-condenser lens (on the TWIN-objective lens) is effectively turned off (negative current reading of ~-90%) to allow for a very small area (0.3-2 nm diameter) of illumination on the specimen. In this mode the illumination is intense and usually convergent on the specimen. This is used for STEM and X-ray analysis. It needs to be noted that, with careful adjustment, it is possible to achieve parallel illumination in the nP mode by focusing the beam from C2 at the front focal plane of the upper pole piece of the objective lens. Because nP allows limiting the illumination to a much smaller circle than the μP, this is a very valuable technique for low-dose cryoEM imaging. See L120C operation.

μP: micro probe mode. This is the normal TEM imaging mode. In this mode the mini-condenser lens (on the TWIN-objective lens - found on all modern Phillipes/FEI/TFS electron microscopes) is turned on (positive current reading of ~90%). A larger area of illumination on the specimen is generated compared to the nP mode. On a two-condenser system, in μP mode, still only one C2 strength setting generates parallel illumination and the illuminated circle is much larger than that from the nP mode.

Acronyms in the Microscope interface
IGP: Ion Getter Pump

TMP: Turbo Molecular Pump

ODP: Oil Diffusion Pump, not on the L120C

PVP: Pre-Vacuum Pump, a rotary vane pump

Pen, Pir: Vacuum gauges, Penning and Pirani.

V: Valves. If a "T" is also drawn on the the double triangle symbol, it is a manual valve. One example for a manual valve is the specimen loading valve, which is open and closed by the turning action during specimen insertion and removal.)

MF: Multi-Function knobs

LTb: The track ball.

pp: Pivot point

LHP, RHP: left-hand side panel, right-hand side panel

HT: High tension, meaning high voltage. This refers to the acceleration voltage (80-120kV for L120C) in the gun. Similarly LT means low tension, low voltage. "LT" can be found on the old carbon evaporator, where it refers to the low voltage, high current power supply for heating the carbon rod to incandescent temperature.

Wobbler
"A wobbler is a mechanism for rapidly switching a microscope element or function from a negative value to an identical but positive value; it can thus be on beam shift or beam tilt, image shift, a stigmator, objective-lens current, high tension, etc., even though the traditional meaning is the beam-tilt aid for focusing the TEM image."

The "Wobbler" button on the right-hand side panel is for turning on "the beam-tilt aid for focusing the TEM image"

The alpha Wobbler (usually assigned to R2) is for rocking the CompuStage.

Spot size
Strength of the condenser lens C1. The term "spot size" refers to the de-magnified gun crossover image on the specimen - the minimum size of the illumination spot. Stronger C1 lens (larger number on the spot size control slider) moves the crossover below C1 up, further away from the C2 lens, resulting in a smaller spot size. With stronger C1/smaller spot size/larger slider number, less electrons will pass through the condenser aperture. This gives direct control on the total electron dose rate on the specimen. From spot size 1 to 11, every stop reduces the dose rate of electron from the illumination system by a factor of 2. If desired, the dose rate can be further reduced by using a smaller C2 aperture.

It needs to be pointed out, that on modern electron microscopes, when the spot size (C1 strength) is changed, the strength of C2 is in fact also changed automatically by the controlling computer to maintain the crossover from C2 at the save position. This way the user is relieved from readjusting the C2 (Intensity).

Intensity
Strength of the condenser lens C2. Turning the Intensity knob clockwise increases the strength of the C2 lens. After passing the crossover point (minimum size of the illuminated circle on the screen, in fact the image of the filament), turning the knob clockwise decreases the intensity in the illuminated area due to the spreading of the illumination beam. However the total dose rate of electron on the specimen remains the same. The total dose rate on specimen should be changed through changing the spot size or the condenser aperture.

Turning the Intensity knob clockwise always increases the strength of the C2 lens. Normally, a cross over should always be present above the objective lens and the specimen. Therefore one should always first find the crossover point then turn the knob clockwise to dilate the illumination circle.

Micro condenser
A lens immediately above the upper pole piece of the objective lens. In fact, it is part of the objective lens assembly. The main function of this lens is to allow a larger area to be illuminated for lower magnification imaging. When the micro condenser lens is activated, the microscope is operating in the micro probe mode. When it is off (its current is reversed, indicated by a negative strength value), the microscope is in nano probe mode.

On a two-condenser system (non-Titan), in the micro probe mode, the parallel illumination beam is about 5x the diameter of the parallel illumination beam obtained in the nano probe mode. That is, ~21 μm with a 150 μm C2 aperture in μP mode versus ~5 μm in nP mode. Consequently, for cryo-EM work, using nano probe mode and obtain parallel illumination becomes necessary to avoid damaging protein in adjacent holes.

Twin lens
A symmetrical objective lens design (Riecke-Ruska type), in which the specimen is placed at the center of the objective lens, with two magnetic fields of equal strength above and below it. A micro-condenser lens is added as part of the objective lens assembly to allow larger area to be illuminated (see above).

A magnetic lens is composed of an upper ring-shaped pole piece and a lower ring-shaped pole piece. A gap between the two rings will have concentrated magnetic field lines and the magnetic field expand towards the center of the bore with decreasing strength. This generates a curved magnetic field in the bore, which acts at a convex lens for electrons (see http://www.feynmanlectures.caltech.edu/II_29.html). The specimen is usually immersed in the magnetic field of the objective lens on electron microscopes. The magnetic field above and below the specimen are called the prefield and postfield.

In the twin lens design, the objective upper and lower pole pieces have the same size and shape, giving a symmetric magnetic field along the optical axis. The specimen is placed at the 1/2 position between the upper and lower pole pieces. The magnetic field above and below the specimen act as two identical lenses. The prefield is also called the objective condenser lens, apparently referring to its effect of acting as a final condenser lens. The postfield can be referred to as the objective imaging lens, as it is the real imaging objective lens.

It is obvious that the upper lens plays an important role in the illumination. Ideally (but not necessarily for non-cryoEM applications), the illumination beam should be set to form a crossover at the frontal focal plane of the upper lens, so that a parallel beam is generated to illuminate the specimen. It is also obvious that since the twin lens is symmetrical, the positions of the frontal focal plane of the upper lens and the back focal plane of the lower lens are both controlled by the Focus knob and will change simultaneously. Therefore when the frontal focal plane of the upper lens is at the right position, the back focal plane must also be at the right height. The "right height" of the back focal plane of the lower lens is the height of the objective aperture in the SA magnification range. When the microscope is properly aligned, pressing the Eucentric' button should bring the objective lens to a preset value, at which the front and back focal planes are both at their respective correct height.

Note: On the Talos L120C, a C-Twin lens is used.

What is in contrast to the symmetric twin lens is the high contrast lens. In the high contrast lens design, the upper and lower pole pieces have different geometric sizes. Usually the upper pole piece has a smaller bore. Consequently the field between the two pole pieces (the magnetic lens) is asymmetric. The specimen plane is designed to be located at the 1/3 position in the pole piece gap, and the back focal plane is at the 2/3 of the position in the pole piece gap. The objective lens is then considered as a single lens below the specimen, even though the specimen is in fact still immersed in the magnetic field of the objective lens. Electrons entering the high contrast lens does not experience a strong field for long before hitting the specimen, therefore the condensing effect of the prefield is not very significant. (Page 199-200, Introduction to Conventional Transmission Electron Microscopy, Marc De Graef and De Graef Marc, Cambridge University Press)

Focus
Strength of the objective lens under imaging mode.

The magnification of the objective lens is in fact a fixed value, errors in it leads to errors in the overall magnification. The image from the objective lens is always at a fixed height (the SA aperture plane) in the column, where the diffraction lens is coupled at. Therefore the specimen should always be brought to the eucentric height to ensure that the specimen-objective lens distance is correct. In addition, achieving parallel illumination is dependent on bringing the illumination beam crossover at the frontal focal plane of the objective lens. Keeping the objective lens current close to the predetermined eucentric value allows keeping the height of the frontal focal plane of the objective lens relatively fixed.

Therefore, the "Focus" knob should only provide the necessary fine-tuning of the focusing.

Eucentric height
"" The α tilt of the CompuStage is constructed in such a way that it is possible to tilt around it without having large apparent movements of the point of interest on the specimen. This is called eucentric tilting and is achieved by bringing the point of interest to the same height (with the Z axis) as the a tilt axis itself : the eucentric height. The eucentric height is important because it not only provides an easy way of tilting without having to correct specimen position continuously, but it also defines the reference point inside the microscope for all alignments, magnifications, camera lengths, and so on. In general one should work at the eucentric height (the only reason for deviating could be that at very high b tilts and specimen positions away from the center, the range of the Z axis may not be sufficient to bring the specimen to the eucentric height).""

The height of the alpha rotation axis of the CompuStage. The optical system of an electron microscope is designed to have the specimen at the eucentric height. If the specimen is too far away from the eucentric height, even though an image can still be formed on the detector by aggressively adjusting the focus, owing to the fact that the magnification is determined essentially by the distance between the objective lens and the specimen, the actual magnification on the screen will have considerable error. The quality of the image may also be affected. Ideally, when the specimen has been brought to the eucentric height, pressing the EUCENTRIC button (which loads the saved objective lens value to the lens) should bring the sample very close to focus.

Parallel illumination
Illuminating the specimen with parallel beam. The specimen is in fact situated between the upper and lower pole pieces of the objective lens, each of which is in fact a lens by itself. When the illumination beam is focused at the front focal plane of the upper pole piece of the objective lens, parallel illumination can be achieved. As a result, if the specimen is crystalline, very sharp diffraction spots can be observed at the back focal plane of the objective lens (= the back focal plane of the lower pole piece), which can be observed in the diffraction mode.

Camera length
The effective length of specimen to the diffraction image plane in diffraction mode. Controlled by the magnification knob in diffraction mode. The original diffraction pattern is formed at the back focal plane of the objective lens, which is only a few millimeters below the sample. In diffraction mode, the projection system projects the diffraction image to the camera with certain amount of magnification. The Camera length displayed at the bottom of the computer screen is the effective length between the specimen and the diffraction image center. Multiplying camera length by the diffraction angle (in radians) gives the spacing of the diffraction spot. For example, if the camera length is 500 mm and the diffraction angle of a spot is 2 mRad, then the diffracted spot should be 1 mm away from the direct beam on the camera.