Instructions for the Philips CM100 TEM and peripherals

Please refer to the Philips CM100 Operating Manual (PW6021) on the shelf for the description of components: Chapter 7, Electrical and Mechanical Controls - Location and Description.

Check-in

enter the In time on the log sheet and check previous entries for unusual remarks
initial setup for the right-hand control panel
- push in PANEL DIM knob to turn on the indicator lights
- UHV and HIVAC should be on, if not STOP
- turn on the Sony menu monitor which should display the TEM BRIGHT FIELD page (if not, check the menu page tree in the Appendix)
  
  Note: Whenever the READY button is lit, you can press it to return to an upper level page.
press MODES (right 1) to access the MODE SELECTION page, then press CONFIGURATION (left 8)
- TUNGSTEN should be selected for filament, if not STOP
- make sure FIL LIMIT is set as posted and not 0, if not STOP
hit READY to back out to the previous page
press MODES (right 1) to go to the MODE SELECTION page then select TEM (right 1) for the TEM BRIGHT FIELD page
set MAGNIFICATION to 1100× and select SPOT 2
press key right 2 for PARAMETERS
make sure EMISSION and HIGH TENSION are set as posted, and HIGH TENSION is ON, if not STOP

Note: Check the Appendix if you want to use a different accelerating voltage.
hit READY to back out to the TEM BRIGHT FIELD page
initial setup for the column
- condenser aperture set as posted and engaged
- objective aperture set as posted (usually 4) and engaged

Specimen loading

DO NOT touch any parts of the specimen holder except the black cap
make sure the goniometer drive is in the locked position
grasp the black cap securely and pull the specimen holder out until it stops

Caution: Place the other hand on the goniometer to catch the holder in case it accidentally slips and is being sucked back in (it is under vacuum!). If that happens, the crystals at the end of the holder and the seat inside the column can be damaged; a rather costly repair and serious down time.
rotate the specimen holder clockwise until the marker is at 5 o'clock position and then ease it out gently to break the vacuum
Caution: It does take a certain amount of force to break the vacuum. If you do it too fast, you are more likely to scrape the holder against the inner surface of the airlock damaging both parts.

Here are 2 ways to do this safely:
- if you have longer fingers, you can grasp the black cap with your thumb, ring and small fingers, then use your index and middle fingers to push against the goniometer gently until the vacuum breaks
- otherwise, you can rest your left hand with the knuckle on the goniometer just adjacent to the z-position thumb screw, then use the thumb, index, and middle finger to push on the cap gently until the vacuum breaks while stabilizing the holder with the right hand
place the specimen holder on the support
steady the holder and lift open the spring loaded clamping device with the pin tool
place the grid in the recess and close the clamp
check for dust, lint, or other debris on the rod; if present, carefully remove them with Ross lens paper
gently re-insert the holder with the marker at 5 o'clock position and make sure that you do not bump the rod against the inside of the chamber, the red airlock indicator light on the specimen chamber should come on to indicate initiation of pre-pumping
fully insert the rod

Note: as soon as a vacuum is formed, the holder will get sucked in further and you should hear a click
when the indicator light goes out (~ 1 min), turn the holder counterclockwise until marker is at 12 o'clock position and guide the holder to slide in slowly; put the other hand on the goniometer to catch the holder in case it accidentally slips and slams in
gently wriggle the holder slightly to make sure that it is all the way in and sits squarely in the chamber
monitor the VACUUM page (right 7) until IGP is ≤ 27 (~ 5 min) before proceed to Filament saturation

Filament saturation

turn the FILAMENT knob clockwise, pause between each click until the on screen message is clear or wait 0.5 s
when you reach the preset limit, the system will beep
check the emission current against the posted parameters, should be < 20 µA

Caution: If current > 20 µA, turn down the filament and notify Facility staff.

Quick quality check (notify Facility staff for any alignment problems)

if possible, locate a hole or area devoid of material in your preparation and then set the magnification to 5800×

bring the beam to crossover with INTENSITY (left-hand control panel), center it with SHIFT XY (right-hand control panel), and perform the following

action	observation	possible problems and remedies
check the exposure time	much longer than the posted reading	gun alignment or filament saturation: do gun tilt procedure in the Appendix, and if needed, do extended procedure for filament saturation in the Appendix
change INTENSITY upon crossover	beam shift (see example in the Appendix)	CONDENSER APERTURE alignment: center according to this procedure in the Appendix

Specimen exchange

do Check-out procedure (see below) steps 1 to 4
perform Specimen loading procedure steps 1 to 6
replace the grid and complete the rest of procedure, IGP should be ≤ 27 before turning on the filament

Setting the eucentric position (essential for using tilt and doing measurements)

center and focus a recognizable object at a magnification one click higher than the max. magnification you will use; however, the practical max. magnification that one can do this procedure is about 10 500×
flip up the goniometer drive to disengage it from the tilt mechanism
rotate the tilt mechanism ± 2 ° to 5 ° upon 0 °, if the object moves sideway about the center, hold on to the tilt mechanism, turn the "Z" position thumb screw on the goniometer below the black cap of the specimen holder a small amount to either direction

Note: apply a smaller tilt angle if the object moves a large distance or off the screen
rotate the tilt mechanism again, if the object shows more lateral movement, turn the thumb screw in the other direction; otherwise, keep adjusting the thumb screw until there is minimal lateral movement upon rotating the tilt mechanism
return the tilt to 0 °, check the focus, repeat the procedure if the focus is too far off
re-engage the goniometer drive

Gatan CCD camera

turn on the power supply
set SHUTTER to AUTO (green LED on)
turn on the Mac with power switch on the keyboard and turn on the monitor, then launch Digital Micrograph
select SSC | camera setup
- exposure time: 0.3 s
- image size: 512×512 pixels (default to 2× Binning, can do 128×128 pixels to 1024×1024 pixels), binning is possible when size is ≤512×512 pixels and will improve response time at the expense of resolution
- configuration: magnification correction is 0.26, and the shutter should be closed
optimize the intensity: focus the beam, center illumination with SHIFT XY
locate object of interest and focus
adjust illumination to get an exposure reading of 5 s, or the shortest available if exposure is longer than 5 s
insert camera and choose under SSC either
- Acquire gain normalized, or
- Gain normalized view ("almost live" view with very slow update, hit space bar to capture image)
retract the camera whenever you are not actively taking a picture
save the images to your directory in the drive "Images", the following formats are most popular
- Gatan: 16-bit, see Appendix for more info
- TIFF, display: 8-bit (usually with scale bar and magnification markers burned in)
- TIFF, original data: 16-bit (better dynamic range than 8-bit; but may require levelling of the histogram, and annotations such as scale bar and magnification marker are not saved), see details in the Appendix

File transfer using niftytelnet/SCP (secure copy)

from the Apple Menu choose niftytelnet, the Connection dialog box should open to allow selection of a file server; if not, do File | New Connection (default is if.biology.washington.edu but choose other as needed)

Note: select "SCP" for secure copy, "Connect" is for ssh
to transfer your files, click the SCP button
click add Files/Folders for file selection, your files should be in "Images", accessible from the Desktop

Note: To transfer folder(s), check the box "Copy Contents of Nested Folders". Putting all of the session's files in 1 folder is the most efficient way.
if applicable, fill in the Destination Path field with the name of your preferred directory on the server
click on Start Copy, enter the username and password when prompted
select File | Quit to exit niftytelnet

Tips for using ssh

enter the username and password when prompted (for Homer or Dante, press s to obtain a shell after you log in)
useful commands for a SSH session
- mkdir dir-name (create a new directory)
- cd dir-name (change the current path)
- pwd (list the current path)
- ls -l (list the files in the current path, long format)
- exit (terminate the session)

Check-out

select magnification 1100×, SPOT 2, make sure both apertures are selected as posted and engaged
turn the FILAMENT knob counterclockwise, pause between each click until the on screen message is clear or wait 0.5 s
when the filament setting is down to 0, the system will beep
wait 10 s
remove your sample and replace the specimen holder in the scope
if you use the camera (check with the next user about leaving the camera and computer on)
- make sure the camera is out and turn off the power supply
- quit Digital Micrograph (File | Quit)
- upload images to a file server using niftytelnet/SCP
- shutdown the computer (Special | Shut down)
return to the TEM BRIGHT FIELD page
pull out PANEL DIM to turn off the display
turn off the Sony menu monitor
enter Out time in the log sheet, check the shutdown procedures listed and complete the log
cleanup the work area before you leave

Appendix

Saturating the tungsten filament

Caution: Do NOT perform these steps unless you have been trained by Facility staff on this procedure.

load the specimen as usual
go to CONFIGURATION page to monitor FILAMENT HEATING
turn the FILAMENT knob clockwise, allowing 0.5 s wait between each click (or until the message is clear) and pause when ACTUAL reaches 14, or 5 below the posted FIL LIMIT, whichever is lower
the emission current meter should read less than 10 µA, otherwise turn down FILAMENT (ACTUAL to 0) and STOP
if there is no beam, check the following:
- if there is no emission current, STOP
- if emission current is ≤ 10 µA
  1. try adjusting INT, it may be near the limits; press RST
  2. beam may be off center, try SHIFT XY
  3. the specimen holder may be blocking the beam because the mechanical stage controls are too far from the mid positions: left is ~10, right is ~0; center the holder
  4. grid bars may be blocking the beam, use the mechanical stage controls to move them out of the way
STOP if there is still no beam, otherwise proceed with a clear area or a hole on your grid
push FIL LIMIT (left 2) to deselect/unlock

Note: You can start from here if the filament desaturates right in the middle of your session.
push ALGN on right-hand control panel
set magnification to 5800×
focus beam with INT and center with SHIFT XY
the beam will probably look irregular with some dark areas resembling an "eye" shape image
turn FILAMENT further clockwise to obtain maximum and even brightness with little or no serration (dark areas)
if emission current is > 20 µA, turn down FILAMENT and STOP; otherwise continue
center beam with SHIFT XY
optimize gun tilt
1. select TILT (right 4) (detailed in Gun TILT adjustment)
2. use MULTIFUNCTION XY to get a symmetrical image with maximum brightness i.e., shortest exposure time
push ALGN to exit back to CONFIGURATION
lock FIL LIMIT (left 2)
scope is now ready
repeat this same procedure if you have more samples
do check-out as usual, but record FIL LIMIT in Remark if it is different from the posted value

Gun TILT adjustment

Caution: Do NOT perform these steps unless you have been trained by Facility staff on this procedure.

locate a hole or an empty area on the grid
bring magnification to 5800×
focus and center beam
check exposure time against posted value
press ALGN on right-hand control panel
press soft key right 4 for gun TILT SHIFT to highlight TILT
use MULTIFUNCTION XY to maximize the brightness (minimize exposure time) and use SHIFT XY to keep the beam centered
if exposure still differ much from posted value, notify Facility staff
press ALGN to exit this procedure

Changing HIGH TENSION

Caution: Do NOT perform these steps unless you have been trained by Facility staff on this procedure.

The filament is usually saturated at 100 kV but you should checked the posted operating parameters. Other kV values can be used, please check with Facility staff.

press soft key right 3 to select desirable HIGH TENSION
saturate the filament; currently, 80 kV and 100 kV use the same FIL LIMIT

Note: If the HIGH TENSION fails to turn on, the Wehnelt s.w. protection may be tripped and requires a reset.
make sure that the emission current does not exceed 20 µA
it may be necessary to correct for objective lens astigmatism (see below)
return the scope to the posted HIGH TENSION setting after your session unless otherwise instructed by Facility staff

Objective lens stigmation for 80 kV and 100 kV

Caution: Do NOT perform these steps unless you have been trained by Facility staff on this procedure.

locate a roundish object at a magnification higher than the max. that you will use
focus beam, adjust for max. and even illumination; overfocus to spread the beam, if needed
focus image
press STIG on right-hand control panel
press soft key left 7 for OBJ
press soft key right 7 for CHANNEL to highlight 1 for the 80 kV preset or 2 for the 100 kV preset
record the preset reading for both A and B
use MULTIFUNCTION XY to correct for any astigmatism and then refocus
repeat the above step until there is no observable astigmatism
if things go south, return to the preset A B reading, and redo the last 2 steps
press STIG to exit this procedure
at the end of your session, restore the preset A B reading

Condenser aperture alignment

Caution: Do NOT perform these steps unless you have been trained by Facility staff on this procedure.

move to a less important part of your prep if you want to minimize beam damage
select low to medium M range magnification
select SPOT 5, center and focus beam

with INTENSITY underfocus and then overfocus the beam to check for lateral shift

	underfocus	crossover	overfocus
aligned
misaligned

use the 2 mechanical adjustment knobs to shift the beam until there is no more lateral displacement going from underfocus to overfocus

it is rare that there is a need for a different aperture size other than 3

Condenser aperture size
position	1	2	3	4
diameter (µm)	100	150	200	300

Objective aperture selection and alignment

Caution: Do NOT perform these steps unless you have been trained by Facility staff on this procedure.

move to a less important part of your prep if you want to minimize beam damage
select medium M range magnification
center and focus beam, adjust for max. and even illumination; overfocus to spread the beam, if needed
focus image
press D on right-hand control panel next to MAGNIFICATION for diffraction mode
you will see a brighter spot centered more or less in the beam on the fluorescent screen
camera length will show on the display in place of magnification
use the MAGNIFICATION knob to change the camera length to 640 mm

carefully rotate the objective aperture holder to the desirable position

objective aperture size
position	1	2	3	4
diameter (µm)	30	40	70	100

use the 2 mechanical adjustment knobs to center the beam with respect to the bright spot

Note: The aperture could be way off the center, simply turn the adjustment knob to move the beam until you can see the central bright spot.
press D again to go back to bright field mode
you should check the stigmation after changing to a different size aperture
change back to position 4 and center the aperture after you are done

Handling 16-bit TIFF

16-bit vs 8-bit
- a 16-bit TIFF will have 2¹⁶ or 65 536 levels of grey scale, a 14-bit image from the camera will have 2¹⁴ or 16 384 levels, but most computer displays are 8-bit with 256 levels
- even with a properly exposed image acquired with the camera, the pixel intensity distribution (as represented by a histogram) will only occupy the lower quarter of the full range of a 16-bit TIFF
- opening this file on most software will only give you a very dark or even black image unless the software remaps the histogram to 8-bit by windowing such as used by ImageJ
- most of the time images that are saved as 16-bit TIFF in Digital Micrograph, adjusted as shown below, and then converted to 8-bit will have better dynamic range than images save as display (8-bit) in Digital Micrograph
convert to 8-bit using Photoshop
1. open the 16-bit TIFF and do a levelling (Image | Adjustments | Levels…)
2. expand the histogram by pulling the white point slider (right triangle) under the histogram to the left until it almost reach the edge of the histogram, click ok
3. do level again, this time you should see the histogram spread over a lot more, move the black point slider (left triangle) and the white point slider to bracket the ends of the histogram
4. moving the midtone slider (center triangle) will adjust the gamma of the picture
5. to convert it to 8-bit, do Image | Mode | 8 Bits/Channel
convert to 8-bit using ImageJ
1. do Image | Adjust | Brightness/Contrast…, or Window/Level… (brightness or level is the offset, contrast or window is the range)
2. bracket the histogram by sliding the Minimum and Maximum, or for Windows/Level adjust both to achieve the same
3. then do Image | Type | 8-bit

Opening images saved in Gatan format for Digital Micrograph 2.5

use ImageJ
1. install the Bio-Formats plugin (loci_tools.jar) to allow drag and drop opening of dm2 files (with or without file extension), or use Fiji which is just ImageJ prepackaged with loads of plugins including Bio-Formats
  
  Caveat: Reading of calibration and other metadata are not supported yet.
2. use File | Import | Raw… with the following parameters
  - Image Type: 16-bit Signed
  - Width and Height: use either 512 or 1024 pixels
  - Offset to First Image: 24 bytes
  - Number of Images: 1
rename the file with extension .dm2 and open it in LISPIX for Windows
use Photoshop, File | Open as…, select Raw and use the same parameters as in ImageJ above, will need to adjust level

Note: If you do a hexdump of the tail of the file, you will find the date and time stamp, the indicated magnification, and many other parameters.

On Screen Measurements

quick size estimation: the 2 concentric marks on the large viewing screen are 40 mm and 5 mm in diameter e.g., at 25 000×, the inner circle indicates 200 nm
use RSET DEFOC for height
1. set eucentric height
2. focus on structure
3. press soft key right 5 for RSET DEFOC
4. focus on substrate
5. defocus displays the height
Note: Do not use the wobbler (WBL) to focus in this procedure, it will set the defocus readout to 0.
use MEASURING for distance with respect to an external reference e.g., the pointer or any screen markings
- single measurement
  1. set eucentric height
  2. focus on structure and align one end to the reference
  3. press soft key right 6 for MEASURING
  4. press soft key right 8 for ENTER
  5. use SHIFT XY to move the structure and align the other end to the reference
  6. d1 displays the distance
  7. press soft key right 8 for ENTER twice to clear d1
- cumulative measurements
  1. do single measurement
  2. use the mechanical specimen translation controls to align one end of the 2nd item to the reference
  3. use SHIFT XY to align the other end of the 2^nd item to the reference
  4. d1 displays the cumulative distance
  5. repeat for more
- comparative measurements
  1. do single measurement
  2. press the soft key right 8 for ENTER to store d1 and activates d2
  3. measure the 2^nd item
  4. display will show d1/d2 and the angle between them
Note: Press READY to exit MEASURING.

Some of the commonly used Menu pages

MICROSCOPE STATUS and other top level pages

TEM BRIGHT FIELD and other children pages
TEM bright field page and other children page