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Compiled by: Julian
Thorpe
| |
Use of the Transmission Electron Microscope
(TEM)
**IMPORTANT**
Please don't use the
below as a substitute for having training with Julian on the TEM, WHICH YOU MUST
HAVE BEFORE USING THE MICROSCOPE(!),but
you may find this useful to refresh your memory after training when you are actually sat at the
TEM.
Please e-mail Julian
or phone 7585 if you would like to arrange a training session with me.
Also, if you have any problems with the TEM, and I am not around
at the time, please mail me with
a description of the problem.
Specimen Rod Removal:
The specimen rod is normally left in the TEM (to keep it clean within
the vacuum). N.B.: During the following, please keep a firm grip of the
specimen rod otherwise the vacuum will tend to suck it back into the column
and could cause expensive damage!! And if you feel any resistance, please do not
use any force(!!) - just come and ask me to help.
To withdraw the specimen rod,
 | Pull specimen rod outwards carefully until it
reaches a natural stop |
| Turn to the right (about 45 degrees) and pull out
further until it reaches another natural stop |
| Then turn to the left (again
about 45 degrees) |
| Now flick the switch (below the specimen airlock) to
air, wait a few seconds (until you hear a click) |
| Withdraw the rod carefully (keeping it in a horizontal plane) from the TEM |
Grid Insertion:
N.B.: During the following, please do not touch the rod at all with your hands
[below the level of the vacuum O-ring]; grease on the rod will lead to
charging of the electron beam and imaging problems
| Place the specimen
rod on the special rod holder and draw back the securing flange by placing
the wooden stick in the hole |
| Now remove the small plate that secures the
grids in place with forceps and place your 2 grids carefully over the grid-holes
(N.B. grid position 1 is that nearest the tip of the holder) |
| Replace the
plate over the grids and carefully slide back the securing flange with
the stick |
| Visually check that the grids are still central within the grid
holes and invert and gently tap the rod to ensure that they are secure
and will not drop out within the TEM!!
|
Specimen Rod Insertion:
| Carefully insert
the rod into the specimen airlock so that the small protruding metal stud
goes into the groove (at 12 o/c) on the outer face of the airlock |
| Hold
in this position and flick the switch to evacuate |
| N.B.: During the
following, please keep a firm grip of the specimen rod otherwise the vacuum will
tend to suck it back into the column and could cause expensive damage!!
And if you feel any resistance, do not use any force!!!! |
| Wait
for the green light to illuminate and then carefully turn the rod to
the right (45 degrees) and carefully allow the rod to slide in until it reaches
a natural stop |
| Now turn to the left (45 degrees) and carefully allow to
move forward completely into the imaging position. |
| You can move from grid 1 to 2 by rotating the knob right at the end of the
specimen rod (fully one way or the other). |
Initial Viewing of Your Sample:
| If the accelerating voltage is not already on (illuminated), press 'acc voltage' ready/off
button (left console of EM) and select the voltage required (usually 100kV). Now turn up
the filament current (just below acc voltage button) quite slowly and fully clockwise. The electron beam is now on. |
| Initial observations are more easily accomplished in 'low mag' mode.
Select the 'low mag' function button (upper right portion of left console)
and then withdraw the objective aperture from the EM. The objective aperture
is at the same level as the specimen rod on the left side of the EM. To
withdraw the aperture swing the rod at the base of the aperture
rod unit from left to right (please do this carefully as it is on a sprung
mechanism). |
N.B. Please note that the specimen rod and the objective aperture assembly
are the only parts of the EM column you should ever need to touch!!
| From hereon, the only controls on the EM console that you need to use
are the following: (left console) 'low mag' and 'zoom' function buttons,
'magnification' and 'brightness' control knobs; (right console) focus 'coarse'
button, 'focus' and 'brightness centring' control buttons. These are all
clearly labelled and easily accessible in the dark when you are familiar
with their position. Please avoid touching anything else. If you
get into trouble please cry for help rather than attempt things which may
result in the need for a major re-alignment of the EM! |
| Now you should see a very low magnification image of your grid; if you
do not then turn the magnification to the lowest mag (fully anticlockwise
until you hear the beeps) and then adjust the brightness so that the electron
beam fills the screen. Now you can look over your sections and select an
area of interest which you should position in the screen centre. |
| Now you need to carefully replace the objective aperture by swinging
the rod to the left and then select the 'zoom' function. If you do not
see anything after this, follow this procedure: select the lowest magnification
and then turn the brightness control knob so that the electron beam fills
the screen. If, after this, you still do not see anything (or at best a
rather dark image) then the objective aperture is probably off-centre. Objective
aperture centring is achieved by x and y shifts on the aperture rod assembly
(these are the smallest knob (furthest from the EM column) and the small
knob on the right of the assembly). Please go to OL
Aperture Alignment for details as to how to do this. |
Routine Imaging:
To achieve a good final image, follow the procedure below:
(i) Lift the central portion of the fluorescent viewing screen (by raising
the large flange to the left of the viewing chamber) so that it is perpendicular
to the binoculars. Slide the binoculars into position and carefully focus
the binoculars themselves onto the small cross in the screen centre.
(ii) Now select the magnification you require and adjust and centre
the beam via the brightness and brightness centring controls.
(iii) Focus on your specimen firstly with the focus 'coarse' button
selected (illuminated). When at best focus, de-select the 'coarse' button
and now you can fine-focus the image.
(iv) At this stage you need to correct for astigmatism in the objective
lens. (Astigmatism is present in any lens whether electromagnetic or glass;
the main symptom of astigmatism is that, as you go through focus, the image
appears rather blurred in one plane when under-focused and in a plane at
180 degrees to this when over-focused. If astigmatism is very bad then
it is impossible to focus the image at all well). Please go to Astigmatism
Correction for full details as to how to do this.
TEM Alignment:
The best possible image will only be achieved when the TEM
is aligned properly. The Hitachi engineers are very good and they leave the
TEM nicely-aligned, in respect of all the more complex alignment procedures,
after every service visit. However, there are a number of basic alignments
that vary with kV, spot size, aperture used, etc. and these are worth
checking when you start to use the microscope and after changing the spot
size. viz.:
High Resolution Imaging:
N.B. For all the below, please ask myself for help if you have not
received training in any particular routine.
There are various factors that will help you get the most out of your sample
imaging. Some of these will be influenced by the type of sample you are
examining (eg. is it electron beam-sensitive, low or high contrast, relatively
dense or thin, etc.). The general rules of thumb are:
|
Higher accelerating voltages (kVs) will provide better
resolution but decreased contrast. |
|
Contrast can be enhanced by using a smaller aperture (3 or
4) but this is at the expense of some resolution. |
| A smaller spot size will provide better resolution but the
electron beam will become dimmer. There are 8 spot sizes, with 1 giving the
brightest, lowest resolution image and 8 the dimmest, highest resolution.
Spot size 6 is a reasonable compromise for most viewing with 4 being better
for higher resolution imaging. Please go to Changing
Spot Size for details as to how to do this. |
Magnification Calibration:
N.B. If you want to obtain precise measurements of your sample, I now have a
calibration sample for higher magnifications on the TEM. This consists of
catalase crystals that have known lattice spacings (of 8.75 and 6.85nm). I can
show you individually how to do this as and when required.
A couple of important points to remember before taking measurements:
| Firstly, turn the magnification knob up to the highest
magnification and then down again. (This standardizes the lenses for effects
of hysteresis). |
| Secondly, the specimen Z height affects the
magnification. If you don't know where or what this is, then I am quite
happy (!) as it routinely doesn't need to be touched. If ever any of you
feel the Z height needs checking or changing, please ask me to do that and
then I can alert everyone via email that the magnification calibration
should be re-checked. |
| Thirdly, I would suggest that if your measurements need to be
absolutely precise, then you should calibrate the magnification before you
start using all the conditions (kV, spot size, etc.) and
magnifications that you will be using to image your sample.. |
| ***Very Important*** Finally, I strongly suggest
that you actually make a note of your own calibration numbers and make sure
they are correct before you start a new session. I can show you how to
check these numbers. In the future I am going to try and sort out a system
whereby we can all have our own individual magnification calibration tables
so that we are all responsible for own measurements! |
Other Useful Features on the
TEM:
Although this is not a PC-driven microscope, the Hitachi-7100
TEM does have a range of microprocessor-controlled options which may be
accessed via the cathode ray tube (CRT) screen at left of the EM column
and a numbered and arrowed keypad set into the (extreme left) desk area
of the EM. The most useful and routinely-used functions are as follows:
(1) Specimen Pre-Irradiation: This is a useful thing to do before
you start examining your sections, especially if you are going to be acquiring
images. Basically the programme scans the electron beam around the specimen
to pre-heat it; in this way subsequent thermal drift in the EM should be minimized.
| Simply key in 20 on the numbered pad and press the INDEX button. |
|
Move the cursor on the CRT over the repeat number and select, say, 3 for
a quick pre-heat or 10 for a thorough pre-heat (each scan takes c.50sec). |
|
Now move the cursor to START and press the DATA button (grey rectangular
button on right). Wait for the pre-irradiation to finish before beginning
to examine your grids! |
(2) Memorizing Specimen Positions:
I would recommend that you
use this feature every time you use the EM. It will save you a great deal
of time and avoid wasting imaging time.
| Key in 2
and press INDEX. |
| Move the cursor over MEMORY POINT or DRIVE POINT (this
toggles between the two by pressing the DATA button) and select MEMORY
POINT. |
| Now press select 00 and press the CLEAR button to remove previously
stored memory points. |
| As you move around the sample and find interesting
areas simply select MEMORY POINT and move the cursor over (P0), then select
a number (up to 19 positions can be memorized) and press the DATA button twice to memories the position. |
| To go back to a memorized position, move the cursor over MEMORY POSITION and toggle to DRIVE POSITION
by pressing the DATA button. |
| Now move the cursor over (P0), select a number
and press the DATA button twice to drive to that position
selected. |
The specimen trace is another useful option (this is in the same
screen as memorizing positions). Move the cursor over TRACE
and toggle to ON by pressing the DATA button. If you are going to systematically
scan a section it is a good idea to got to LOW MAG mode and move around
the selected section to outline it; then when you got to higher mags (ZOOM
function) you can scan left to right and down, etc. and see where you have
been over the section.
(3) Image Rotation:
This facility is available up to a magnification
of 50,000X and is a very useful way to frame your image optimally. Simply
rotate the IMAGE ROTATION knob just below the CRT. N.B. Remember
to focus your image after you have rotated the image; rotation may alter
focus and/or astigmatism slightly.
You should now be able to achieve good final images which can be stored digitally
TEM Troubleshooting
(for camera troubleshooting, click here):
N.B. In all cases of problems with the
TEM, please ask me for help if I am around. If I am not around at the time, and
you cannot rectify the problem by reading the below, please mail
me with a description of the problem.
(1) Losing the
beam/image:
| Firstly, check that the voltage to
the electron gun has not been lost. The 'Acc voltage' button on the left
console of the TEM should be illuminated. If it is not, press the 'acc voltage' ready/off
button (just below the voltage buttons left console of EM) and then select the voltage required (usually 100kV). Now turn up
the filament current (just below acc voltage button) as usual, i.e. quite slowly and fully clockwise. The electron beam
should now be on. |
| Second Look at the 'HV Beam' current
monitor on the upper left console of the TEM. The 'standing current' (i.e.
with the filament not turned up via the filament control being turned fully
clockwise) is 100. When you turn the filament up, this should increase to c.
125 on the HV Beam monitor. If this does not happen, and the voltage is on
(see above), then the filament needs replacing. |
| If this happens whilst changing your
grids or changing the objective lens aperture, for example, firstly make sure
that it is simply not a problem with aperture alignment or grid positioning
(over a grid bar or at the end of a specimen shift). The best thing to do
initially is to go to 'low mag' mode and remove the objective lens aperture
and see if you can image the whole grid. |
| However, if this happens suddenly as you are
viewing your specimen, it may well be due to an intermittent alignment fault
with the TEM. If so,
please do the following: PLEASE DON'T try to twiddle
lots of knobs to find the beam again. Instead,
turn down the filament current and turn off the accelerating voltage. Now press the RESET button below the CRT screen on
the TEM left upper console. Turn on the accelerating voltage and turn up the
filament current and you should find that everything is fine again. |
| If this latter does not work,
please do the following: Locate the 'Gun Tilt' (NOT TRANS) X and Y buttons
that you can access behind the swing
down panel lowermost left of TEM console (where the astigmatism correction
knobs are located). Go to 'low mag' mode and remove the objective lens
aperture and also remove the specimen rod to the 'park' position (pull out
and then right). Now turn the X button fully counterclockwise, then sweep
the Y button fully and slowly from left to right (or right to left), whilst
constantly viewing the fluorescent screen (with all the room lights and PC
monitor off, door closed as well is best). Then turn the X button a few mm
clockwise and sweep through the Y button again. Keep on doing this until you
should eventually see the beam. Once found, adjust both the X and Y buttons
to acquire the brightest beam. Reintroduce your specimen and all should be
well! |
(2)
Beam appears dimmer than usual
| If the beam appears dim on first
viewing, or suddenly dims as you are viewing, it may well be that the
filament is ageing and has thus become misaligned. To align the filament,
adjust the 'Gun Tilt' X and Y buttons to and fro in turn until you get the
brightest beam possible. The Gun Tilt buttons are located behind the swing
down panel lowermost left of TEM console (where the astigmatism correction
knobs are located). |
| Otherwise, check that the objective
lens aperture is properly aligned and that the beam is centred, of
course, via the 'brightness centring' knobs on the right console of the
TEM. |
(3)
Loss of movement in the X and Y specimen shifts of the TEM
| If you find that the X and Y
specimen shifts of the TEM suddenly stop working, this can usually be
rectified by pushing in the recessed 'reset' button (with the wooden stick
or the straight [not the delicate curved!!] forceps) that is located just to
the left of the CRT screen displaying the image position, etc. (and just
below the X and Y position numbers) on the upper left panel of the TEM as
you are sat at it. |
|
