ELECTRON MICR OS ING N CO N PY CA S and X-RAY MICROANALYSIS

Dick Briggs John Brady Bob Newton '2000

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TABLE OF CONTENTS

Section 1 Scanning Electron Microscope Operation Specimen Exchange...........................................................4 Generation of the Electron Probe............................................6 New Alignment Procedure ...................................................8 Viewing the Specimen ........................................................9 Screen Text................................................................... 10 Photography ................................................................. 11 Finishing Up................................................................. 13 Section 2 Energy Dispersive X-Ray Microanalysis Theory ........................................................................ 15 Oxford Link ISIS............................................................ 21 Introduction.............................................................. 21 Microscope Setup....................................................... 21 ISIS Startup ............................................................. 22 Image Acquisition....................................................... 22 Beam Current Setup .................................................... 23 X-ray Spectrum Acquisition........................................... 23 Identifying the Elements Present...................................... 24 Quantitative Analysis ................................................... 25 Quant Calibration ....................................................... 25 Advanced Features...................................................... 26 Section 3 Appendices Appendix A: Sputter Coater ............................................... 28 Appendix B: Condenser Lens Setting.................................... 29 Appendix C: Resolution vs. Magnification.............................. 31 Appendix D: Specimen Preparation ...................................... 32 Appendix E: Vacuum Evaporator......................................... 34 Appendix F: Image Manipulations and Labelling Using Photoshop........................................... 38 Appendix G: Using Photoshop 4.0 to Label Scanning Electron Micrographs ...................................... 42

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Section 1

Scanning Electron Microscope Operation JEOL JSM 6400

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SPECIMEN EXCHANGE
It is easiest and most convenient to insert your specimen into the microscope as the very first step. Once it is in, you can generate the beam, make the appropriate adjustments, and begin viewing. When you are finished, you must remove the specimen as part of shutting down for the day. SPECIMEN INSERTION 1. Insert the specimen, mounted on a 12 mm. diameter support stub, into the cylinder of the specimen holder. There is a height adjustment screw in the bottom of the holder to adjust the specimen surface flush with the top of the cylinder. Fix the specimen in place with the set screw. Wear gloves at this step to avoid contamination of the brass holder. 2. Make sure the filament emission is off (knob at right on 2nd control panel fully counterclockwise) and the accelerating voltage is turned off. To do this, push and release the ACCEL VOLTAGE button on the same control panel (the button lamp goes out when it is off).

Working Distance Selector

Secondary Electron Detector Stage Controls: Tilt Rotate X axis Y axis Vacuum Control Button Exchange Chamber Specimen Exchange Rod Specimen Exchange Chamber Isolation Valve

Working Distance Indicator Lamp

General View of the Column

3. Make sure the stage controls are set as follows: X control 25 mm Y control 35 mm Tilt control 0 Rotation control 0° WD control 39 mm This will insure that the entering specimen holder will be aligned with the specimen stage. 4

Attach the rod to the specimen holder by screwing it into the hole. 7. and press the vacuum control button. fully counterclockwise. Then open the specimen exchange chamber isolation valve by turning it fully (90°) counterclockwise (toward you) and pulling it fully to the right. Then place the whole assembly into the exchange chamber. 5 . Then place the whole assembly into the exchange chamber. The light inside the column will go on. 6. SLOWLY) and the ACCELerating VOLTAGE button is off (button light out). The lamp in the control button will go out when the evacuation is complete about one minute. directing the tip into the hole in the side of the specimen holder.] Fully withdraw the rod and attached holder. While viewing the chamber through the glass disk on the exchange rod. carefully slide the rod into the chamber. this will open the valve and start the evacuation of the chamber. Pull the exchange rod back so the rod is caught in the detent of the glass disk. 1. Attach the specimen holder to the specimen exchange knob.JEOL JSM 6400 4. hold it firmly in place. Remove the rod and place it on the stand. so only a skeletonized procedure is given. Do not try to line them up by twisting the rod to one side or the other adjust the positions instead. THE ROD MUST BE SLID IN AND OUT CAREFULLY. close the isolation valve by pushing it in fully and locking it by turning the knob clockwise (away from you)90°. this will open the valve and start the evacuation of the chamber. and the button will light up. Make sure the FILAMENT EMISSION is off (knob at right end of 2nd control panel. [If step 1 above has not been followed. 5. and press the vacuum control button. PERFORM THESE STEPS CAREFULLY. Pull the exchange rod back so the rod is caught in the detent of the glass disk. The lamp in the control button will go out when the evacuation is complete about one minute. While supporting the rod with your left hand near the glass disk. again depress the vacuum control button to vent the exchange chamber. Once the rod is retracted fully. IF THERE IS MUCH LATERAL TENSION APPLIED TO IT. hold it firmly in place. Then open the specimen exchange chamber isolation valve by turning it fully (90°) counterclockwise (toward you) and pulling it fully to the right. THERE WILL BE A LEAK OF AIR THROUGH THE "O" RING IN THE GLASS DISK AND THE VACUUM WILL BE DUMPED. Make sure the stage controls are set as follows: X control 25 mm Y control 35 mm Tilt control 0 Rotation control 0° WD control 39 mm 2. SPECIMEN REMOVAL This is basically the same as insertion. the rod and the hole will not line up. the rod will come loose in your hand. The light inside the column will go on. if not already done. fit the specimen holder into the dovetail slide of the mount on the specimen stage by pushing the specimen holder with the rod. Then unscrew the rod and fully retract it. This is accomplished by screwing the rod into the threaded hole in the side of the holder. The vacuum will be broken. While viewing the chamber through the glass disk on the exchange rod.

Be sure there is a specimen in the chamber. GENERATION OF THE ELECTRON PROBE 1. using the contrast knob on panel 1 Set to around 225 with the brightness knob on panel 1. PROCEDURE #1: ELECTRONIC 4. You will need to make some adjustments: ACC VOLT Set this to 5 kv. Then press PF-2 followed by the number 1. this gives you a screen titled EOS-1 showing most of the important operating parameters. 2. titled EOS-1. Choose one or the other. this will put the cursor on the upper left corner of the right viewing screen. PROCEDURE #2: MANUAL 5. turn these fully clockwise). then hit 1. this is done by turning the knob found immediately to the right of the ACCEL VOLTAGE button. while firmly supporting the specimen exchange rod. this will display information on the right hand screen. it is changed by turning the probe current knob on control panel 1. On the keyboard. Then press the letter L. and remove the rod and holder from the chamber when it comes to atmosphere. Go to the keyboard and press ESC. If it reads something else.JEOL JSM 6400 3. There are now two different approaches to setting the operating parameters: electronic and manual. on the keyboard press PF2. This should be set to 7. vent the chamber by pressing the red button. press PF-5. There should be a list of information on the right screen. 3. Set to around 225. Then press BREAK. This automatically sets the start-up parameters. CL COARSE MAG SEI (two readings) CONTrast BRIGHTness 6 . There may not be anything on the left screen. Turn up fully the brightness and contrast knobs of the right and left viewing CRTs (each has two knobs under the ledge beneath them. Use the arrows to move the cursor around until STARTUP is highlited. 4. This should give you a list on the right of a number of operating parameters and their settings. Close the isolation valve and lock it. Place the rod in its rack and remove your specimen by undoing the set screw. Set this to around 100 with the magnificationknob on panel 1.

with peaks and valleys. If the line moves to the top of the screen while increasing the emission.e. press the button and it will go out. Then go to the GUN ALIGNMENT region of the panel. Using the two knobs on either side of this button. You are heading for a setting where no more turning of the knob will increase the number of electrons emitted. Then adjust the amplitude (contrast) of the line so that it is about 2-3 cm. SATURATION BRIGHTNESS or VERTICAL POSITION OF LINE ON SCREEN DIST ANCE KNOB IS T URNED 8. press the PIC button. As you turn up the knob.JEOL JSM 6400 6. This should give you a full screen (left) image of your specimen. Position the line on the screen using the BRIGHTNESS knob so that it is about midscreen. the valleys are dark areas. using the CONTRAST knob. press it again. if it is. Return to panel 1 and. 7 . 9. Additionally. it should move vertically on the viewing screen. You will want to adjust the brightness and contrast for your viewing pleasure. If not. Make sure the SHIFT button light is NOT lit. adjust the scan line for maximum detail. just below and to the right of the right viewing screen. 10. See the drawing below for a graphic representation of this. simply lower the line using the BRIGHTNESS knob and continue with the emission increase. and perhaps again. this position is known as SATURATION. The overall position of the line on the screen is a function of BRIGHTNESS. As you turn the knob clockwise.. On control panel 1. press the MODE button. You should leave the EMISSION knob set just at this plateau (saturation) position. saturation) on the left screen. in the SCAN box on the left. make sure the scan line is at its maximum height (i. the light should light up. This line should be positioned about midscreen and can be adjusted vertically with the brightness knob. the line will move up a bit. 7. The distance from the top of the peaks to the bottom of the valleys represents CONTRAST. drop down and then move back up and plateau (saturation). Press the ACCEL VOLTAGE button. You should get a bright scan line across the left hand CRT. the peaks themselves are very bright. the scan line on the screen should become uneven. SLOWLY turn up the EMISSION knob. Using the FOCUS knob. under SCAN.

When you change accelerating voltage. Turn PROBE CURRENT up to 13. and the screen sometimes goes blank when changing probe current. 2. it makes saturation tougher. Go to PICT mode. 3. toggle cursor over to SEM-1 and hit enter (return). if it is off. Be sure the instrument is in MODE or SEM-1 position. if you lose brightness. Return PROBE CURRENT to 13 (SHIFT light OFF). If the shift is greater than that. To do this. bring back with SHIFTs (SHIFT light ON). using the traditional horizontal line scan in MODE. press MODE to get the cross-hairs on the screen and superimpose them on the object. if not already there. When finished. Remember. the above procedure must be repeated. This shuts off the upper deflection coils. and maximize brightness using the TILT controls (SHIFT lamp UNLIT). either press MODE on the EOS panel (under the left monitor) or go to PF-2 (EOS-? then page 2. resaturate and then repeat steps 2 through 6. and stepwise drop PROBE CURRENT to 1. Previously. it also needs to be checked when changing the KV. be sure SHIFT light is on). leaving lots of room for error. the beam alignment is off and steps 1 through 6 should be repeated more carefully. return the EOS panel button to SEM. 4. 1994 The recent installation of the BBD (Beam Blanking Device) has necessitated a more thorough and rigorous beam alignment procedure. 1. As you run through the PROBE CURRENT settings from 13 to 1. 5. Obtain maximum signal with SHIFTs at PROBE CURRENT 1(again. this returns the image shift capability if you want it. 6. and maximize signal using TILT Recheck saturation. the image shift should be less than one micron. 8 . If not properly done. and so to get the beam directed straight down this narrow channel is trickier. the ones that allow you to electronically shift the image around. the beam was coming through a large aperture at the top of the column. Turn on the KV and saturate the filament. also be sure to have the scale marker (micron bar) showing. Check what you have done by focusing on some small object at 5000x. but now there is a 50 micra aperture at the very top of the column. including finding the beam when starting up.JEOL JSM 6400 NEW ALIGNMENT PROCEDURE FOR SEM October 10. or simply watching the beam current meter. either by eye in the PICT mode.

You will need to focus the image. it is impossible to get a good clear image. Additionally you can change the working distance by turning the big knob on top of the specimen chamber door. You can move the specimen in a number of ways. 4. etc. 6.e. all the way. press the waveform monitor (WFM) button on panel 1. This is especially important for taking pictures. the image blurs by defocusing. directional blurring as you pass from over to under focus and back. 9 . it may be desirable to make a number of adjustments to the image on the left screen. parallel lines positioned on the screen and superimposed on the line scan. If you adjust the line scan .e. A fine focusing must be accompanied by a correction of the astigmatism. using the knob to the right of the ACCEL VOLTAGE button and reading the setting on the right viewing screen (ACC VOLT). Astigmatism is quite unobtrusive at magnifications less than 1000x. again with the knobs on the specimen chamber. You should see a series of 6 horizontal. These are presented in no particular order. the bright portions of the specimen) up to the second line from the top. 2. In other words.e. Thus there may in this case be no signal reaching the black level. you see that you can change it with reference to these lines. To correct astigmatism. the bottom is full black. 5. Then select another voltage. If astigmatism is present.. i. For taking pictures. the dark areas of the specimen) about halfway between the second and third lines from the bottom. Determine which part of the image should be in medium gray. the signal from the flat "background" should be set at gray. 1. When the specimen has a relatively flat smooth surface with small detail. Then resaturate the filament. 3. You can read the working distance in mm from the right screen or in the information under your picture or from the number on the knob. using the line scan image. get the image in focus and check for astigmatism. Set that part roughly over the third line. depending on the specimen. To properly adjust the SEI (signal) brightness and contrast. You should adjust the viewing CRT contrast and brightness to suit your needs. first desaturate the filament by turning down the EMISSION knob. but there is no directionality to the blurring. and the direction of blurring changes when going from underfocus to overfocus (the directions will be at right angles to each other). Under conditions of astigmatism.JEOL JSM 6400 VIEWING THE SPECIMEN At this time. then there is astigmatism present in the electron probe system. You can ROTATE the specimen or TILT it. The top line represents full white. This may require some judgement and adjustment. When there is no astigmatism. The image must be in focus for the information on the screens to be accurate. Repeat the test for astigmatism and recorrect if necessary. they represent reference lines to enable the user to reproducible set the same contrast and brightness. You can translocate left to right and front to back using the X and Y knobs on the front of the specimen chamber.again by using the BRIGHTNESS and CONTRAST knobs on panel 1. You may want to change the accelerating voltage.. the third line is a medium gray shade.. Use the FOCUS knob. Turn up the EMISSION SLOWLY. Do this SLOWLY. and the valleys (i. To do this. try to get the peaks (i. Look at the image of the specimen on the viewing CRT carefully before you adjust the WFM. note too that there is a FINE button adjacent to it. adjust the STIGMATOR X and Y knobs to obtain the sharpest possible image. Depress this button (it lights up) to allow fine focus. If the image shows a directional blurring during focusing.

1 screen has OPTimum APERTure written on it. Changing the millimicron number will change the magnification). Changing apertures requires a recentering of the aperture. Be sure to have the beam off and the stage positioned properly. based on accelerating voltage and probe current. The magnification and accelerating voltage may be changed this way. To change the position of the text at the bottom of the screen (as a whole block). This is accomplished by the PROBE CURRENT knob on panel 1. Do not undertake this unless you have been shown how to do it. press ENTER key (curved arrow. Type text . 5. then specimen insertion. SCREEN TEXT 1. for the proper diameter final aperture. place cursor on area and press RETURN. but changing the WD simply changes the focal length of the final lens. Place cursor on the block of text. This necessitates amplification. JEOL 1. Use the arrows on the keyboard to move the cursor around on the screen. put the cursor on it and move it up or down with the arrows. To do this. Typing numbers in the other blocks will change the operating parameters of the instrument. follow the instructions in the section on specimen exchange. you may want to change the diameter of the beam or probe. To change the existing text at the bottom of the image: a. This toggles the big cursor on and off with the small cursor used for text input. b. To delete or return text at bottom of screen: a. You may want to change specimens. slow down the scan speed. Press the ENTER key . As you change magnification. This is selected on the right side of the column. and the probe current can be read on the right viewing CRT on the CL COARSE line. To move the cursor: a. You will notice that the bottom line of the EOS . lower right) and it will disappear. lower right corner of keyboard b. This is followed by a number. ESC on the keyboard toggles the cursor back and forth between screens. To write additional material on the screen: a. To diminish this. To return the text. Place cursor on JEOL 2.up to six characters (if you change your mind. INS on the keyboard 3. 4. 3. which introduces noise. 8. 2. Start with the section on specimen removal. CLS will remove the INS effect) 4. 10 . Move this cursor with the arrows to the desired location. 9. using ESC. b. b. This is the instrument recommendation.curved arrow. type in the text.JEOL JSM 6400 7. throwing the image out of focus. As you increase the probe current. Get the cursor on the left screen. Ask for assistance if you think this is necessary. seen as sparkles on the viewing screen. A smaller probe (a higher probe current setting) increases resolution but decreases the amount of signal generated. then hit the BREAK key. you are decreasing the diameter of the probe by changing the amount of demagnification carried out by the condenser lenses.

Handling the film is not difficult. Type 52 delivers a positive print only. panchromatic films. To obtain the latter. If you want your text plus the information across the bottom of the screen. Follow the instructions above regarding focus. Insert the film into the camera (see below). staten magnification. enter your text. d. The instrument is the equivalent of parfocal on the way down: if you are in focus at a high magnification.IF YOU DON'T CHANGE ANYTHING ELSE. Press the D-MAG button within the DISPLAY square on the control panel 1. 4 inches x 5 inches. Focus the image carefully by going to a higher magnification and. b.or which side should face the lens. 3. view it on a reduced screen. Turn off the WFM. 11 . check to see that the image is what you want. to make an exposure you proceed as follows: a. press the SCAN PIC button one or more times. PHOTOGRAPHY INSTRUMENT SETUP 1. you will be in focus at a lower magnification . This latter type is generally more useful. as it gives a negative suitable for further enlargement in the darkroom. and only marginally more expensive. This separates two rollers inside so the film can be slipped in between. Sit very quietly while the exposure is being made. This displays the the waveform of the video signal and also the six parallel horizontal lines. Use the CLS key to erase the text. Both are fine grain.JEOL JSM 6400 c. 2. Type 55 delivers both the positive and the negative. (The regular viewing mode is twice the indicated magnification. Find and center the portion of the specimen you want to photograph. Adjust the contrast and brightness on the control panel so that the peaks are at the second line down from the top and the valleys are between the second and third lines up from the bottom. the end with the metal strip in first. This reduced the image to the actual. This will begin the super slow scan for taking the picture. pushing it all the way in until a sharp "click" is heard Do not squeeze the film in the marked area. press LEFT. Slide the film between the lips on the front edge of the camera. PHOTOGRAPHY 1. The camera we use with this instrument uses two different types of Polaroid film. Press the LEFT button. then hit the BREAK key. brighter image. astigmatism. This gives you a smaller. Return to the appropriate magnification. if desired. 2. Then pull the cardboard cover back out as far as it will go. leaving the film exposed inside the camera. setting up brightness and contrast. 3. Examine the film to be sure which side is up . c. and in the PHOTO area. the same size as it will appear on the photograph. In both cases. See VIEWING section for further details.) Then press the WFM button immediately to its left. Be sure to check for and accommodate any astigmatism that may be present. Make sure the lever on the front of the Model 545 film back is pushed to the right (L) position. Vibration of the column at this point will show up dramatically on your image.

Be sure the f-stop setting is at f 5. NOTE: Dispose of the film pack and the used up coaters properly. Use the coaters supplied with the film. Do this on a paper towel. Inside the cylinder is a plastic holder with a pink sponge attached. etc.ALL THE WAY. Open the pack and separate the print from the negative carefully and quickly. not on the console of the instrument! c. this is saturated with acetic acid and other materials. IMPORTANT NOTE IF YOU ARE PROCESSING NEGATIVES IN THE SINK. Allow the film to develop for 20 seconds.6. and then very smoothly and evenly pull the film out of the camera. IT IS RATHER INSIDIOUS AND SEEMS TO BE ABLE TO GET EVERYWHERE . ETC. covering the entire surface. RINSE EVERYTHING WITH LOTS OF WATER: TANKS. 1. 2. CHECK THE FLOORS FOR DRIPS. Failure to do this will result in faded pictures. Dispose of the film pack and used up coaters properly. BE CAREFUL NOT TO TOUCH THE SURFACE OF THE NEGATIVE. allow it to develop for 15 seconds. slime) to drop off. HANDS. WHEN YOU ARE FINISHED. b. SINK. COUNTER. DOORKNOBS AND ON THE INSTRUMENT. so be cautious. b. Be sure the f-stop setting is at f 16. WHEN IT DRIES.e. They can stain clothes. Rinse at least five minutes in running water. Do this on a paper towel. The recording is finished when the viewing CRT goes back to its rapid scan mode. The print should be coated as soon as possible. Handle carefully and wash any off your skin. Coat the print with the special coaters provided. c. IT TURNS INTO A WHITE POWDER WHICH FLAKES OFF AND MIGRATES EVEN FURTHER. LIGHT SWITCHES. BE VERY CAREFUL ABOUT THE SULFITE SOLUTION. Timing starts as soon as the film leaves the camera. dip in Photoflo and hang up to dry. DRY THE SINK AND COUNTER WITH PAPER TOWELS. TYPE 55 a. Detailed instructions accompany each film pack.INCLUDING FLOOR. Then push the cardboard sleeve back into the camera . 3. c. and it can burn sensitive skin. not on the console of the instrument! 4. Pull the lever to the left. Coat the picture using several strokes.JEOL JSM 6400 d.. BOTTLES. The processing differs with the two different film types: TYPE 52 a. After you remove the film from the camera. Swirl it gently in an 18% solution of sodium sulfite to clear the dye and to cause the developer layer (i. 4. Handle the negative carefully for the emulsion is soft and easily damaged. 12 . The coating material smells. Remove the tabs from both ends of the negative by folding them over. Discard the tabs. creasing and then carefully tearing them away. then peel off the paper covers.

Choose one or the other. The screen should now read EOS. Accelerating voltage at 5 kv b. Then press the letter L. PROCEDURE #1: ELECTRONIC 3. reading their valuse on the right screen: a. Then press BREAK which makes the information disappear and the cursor go to the upper left corner of the right screen. a. There are two ways to do this: electronic and manual. b. there are several steps to take prior to leaving the instrument. Then press ESC and the cursor should disappear. recording all the requested data. If you have been fixing negatives in the sink with the sulfite solution. d. be sure to rinse and dry the sink and counter area. Turn down the contrast and brightness to about 100 5. 6. When you are finished viewing for any period of time. Turn the MAG up to 300. press break and the information will disappear. Remove your specimen from the microscope following the instructions given in the Specimen Exchange section. Be sure the stage controls are as follows: X control 25 mm Y control 35 mm Tilt control 0° Rotation control 0° Working distance 39 mm 2. 4. Take all your stuff and put it away. Sign out in the log book. the cursor will be there. Be sure to return the electronics of the microscope to the proper shut-down positions.1. Go to the keyboard and press PF-2 and then 1. SLOWLY turn the filament emission down. PROCEDURE #2: MANUAL 4. Return the probe current to 7 c. Set the following parameters. indicating it is off. look for spills and drops on the floor. press PF-1. the light will go out. 13 . Push the ACCEL VOLTAGE button and release. all the way.000x. Go to the keyboard and press PF-5. move the cursor down to highlight SHUTDOWN. 3. Be sure to leave the room clean and tidy. At the keyboard.JEOL JSM 6400 FINISHING UP 1. If there is information on the left screen.

Oxford Link ISIS 300 Section 2 Energy Dispersive X-Ray Microanalysis 14 .

Characteristic X-Ray Emission Electron-Sample Interactions Electrons are accelerated through the microscope column.Energy Dispersive X-ray Microanalysis Theory Fundamental Concepts X-rays are generated as a result of the ejection of an inner level electron (low energy) by an energetic electron from an electron column. the energy distribution of the emitted x-rays is continuous. This energy may be emitted in the form of x-radiation called bremsstrahlung (German for "braking radiation"). Basically it is a single 3mm thick by 15 mm2 crystal of silicon which has been treated with lithium (lithiumdrifted silicon). The ejected electron is replaced by an electron from a higher energy shell.1). An X-ray photon first creates a charge pulse in a semiconductor detector. Energy dispersive systems use a semiconductor detector first developed in the 1960's. Because the atomic structure of each element is different. it follows that each element will emit a different pattern of x-rays. acquiring kinetic energy which is transferred to the sample. Since the primary electron can give up any amount of its energy. thus giving up some or all of its energy (Figure 2. X-Ray Continuum (bremsstrahlung) The primary electron may be scattered inelastically by the coulomb field of an atomic nucleus. 15 . the charge pulse is then converted into a voltage pulse whose amplitude reflects the energy level of the detected x-ray. The Oxford system uses the energy dispersive method. The dissipation of this enegy yields a variety of signals. As a consequence even pure elements emit x-rays at many energies.4 λ= or Ε Ε where λ = wavelength of the radiation in angstroms c = speed of light h = Planck's constant E = Energy of the radiation measured in kilo-electron volts λ= These x-rays can be analyzed either by wavelength dispersive methods or energy dispersive methods. The energy lost as it moves from a high energy shell to a low energy shell is released in the form of x-rays. Each element has many energy levels and therefore many potential vacancy-filling mechanisms. Counting over some time. thus the term x-ray continuum. This voltage pulse is converted into a digital signal which causes one count to be added to the corresponding voltage channel of a multichannel analyzer. results in an x-ray spectrum. Planck's equation hc 12.

16 .Energy Dispersive X-ray Microanalysis Bremsstrahlung e Elastically scattered electron Inelastically scattered electron Figure 2.1.2 The incident electron beam can eject an electron from an inner shell. The energy released due to inelastic scattering of the primary electron beam as it interacts with atoms in the sample results in the continuum radiation or Bremsstrahlung e Characteristic x-ray High-energy secondary electron Inelastically scattered electron Auger electron Figure 2. The energy released is given off in the form of characteristic x-rays or it causes the ejection of another electron (Auger electron). The vacancy created may be filled by an electron from a higher energy shell.

K lines γ L lines β α β α K L M N α M line Nomenclature Figure 2. this energy is given off in the form of x-rays.2). The energy of the radiation uniquely indicates the element from which it came. they can only escape from the sample if they are created near the surface. If the ejected electron was weakly bound. Characteristic X-Rays When an electron is ejected from an inner atomic shell by interaction with a high-energy electron beam. The most likely process in most cases is a series of transformations in each of which an electron from an outer shell "drops" into a vacancy in an inner shell. Since they have little energy. X-ray lines are referred to using a nomenclature shown in Figure 2. the (quantum) difference in energy between the vacant shell and the shell contributing the electron.3 Origin of the nomenclature associated with the x-ray lines The intensity of the characteristic radiation under given excitation conditions is influenced by three factors: 17 . namely. Through a relaxation process this excited ion gives up energy to return to a normal ground state. Each drop results in the loss of a specific amount of energy. For instance. For the high energy inner shells. it typically emerges with only a few eVs (<50) of energy and is called a secondary electron. ejecting it with some amount of kinetic energy. hence the term characteristic x-rays. if a collision with a column electron causes an initial vacancy in the K shell and that vacancy is filled from the L shell than the x-ray line is referred to as a Kα peak. the result is an ion in an excited state (Figure 2.Energy Dispersive X-ray Microanalysis Secondary Electrons The primary electron may interact with an electron in the sample. The position of the initial vacancy determines the first letter and the source of the vacancy filling electron determines the second term.3.

it may be scattered in any direction with little loss of energy. thus stimulating a lower-energy emission characteristic of the second element. The total charge conducted (integrate with respect to time) is directly proportional to the energy of the absorbed x-ray. a high-energy x-ray characteristic of element A may be absorbed by an atom of element B. the energy of the silicon Kα x-ray. Secondary fluorescence. but more efficient excitation on the other.5 to 3. Basically an x-ray enters the crystal disrupting the electron structure which in turn causes a temporary increase in its electrical conductivity. This xray could escape from the crystal without adding to the charge pulse in the detector. These backscattered electrons are much more energetic than secondary electrons and so may escape from a greater depth within the sample. The energy measured for the detected x-ray will therefore be less than the actual x-ray energy. 2. The probability that the emitted characteristic x-rays will be adsorbed before they emerge from the sample.74 keV. The result is degradation in resolution on the one hand. The average atomic number of the sample affects the energy lost due to other scattering processes. The presence of elements A and B in the same sample will therefore increase the intensity of characteristic emission from element B and decrease it from A (matrix effect). It is also possible for the incoming x-ray to stimulate the emission of an x-ray characteristic of silicon. It is generally accepted that this trade-off is optimized at an overvoltage (the ratio of the accelerating voltage to the energy of the excited line) of 2. Emitting atom atomic number determines both the ionization cross section and the fluorescent yield. Backscattered Electrons If the primary electron interacts with the nucleus of a sample atom.Energy Dispersive X-ray Microanalysis 1. The amount of this difference will be 1. They don't carry the topographic information of the secondary electrons but the main influence on the strength of the backscattered signal is the mean atomic number within the interaction volume. The higher the atomic number of an atom the more protons present and the higher the backscattered signal. 3. 18 .74 keV below that of the parent peak. Therefore as counts accumulate in an x-ray peak for any major constituent of the sample. Escape Peaks Emitted x-rays are measured by the lithium-drifted silicon detector.allowing them to be detected. which may be one result of absorption. an escape peak can be expected to appear at an energy 1. Effect of Accelerating Voltage The accelerating voltage used in the electron column of the microscope influences both the spatial resolution of the x-ray signal and the efficiency with which characteristic x-rays are excited from the sample. Some are directed back out of the sample. For example. The atomic number of both the emitting atom and the average atomic number of the bulk sample. Higher voltages produce higher energy electrons that penetrate more deeply into the sample and spread more widely than low-energy electrons.

However at 20 KV the ratio of the line intensities changes dramatically as the K lines become more intense than the L lines. The standard operating voltage for EDS analysis on the Oxford is 20 kV. There are a number of statistical considerations that are important to obtaining good chemical analyses. which we hope is very close to E. which is created by counting a number of x-ray photons of various energies.Energy Dispersive X-ray Microanalysis Relative peak intensities will change as the accelerating voltage is changed. The first type of error is classified as systematic error. The second type of error is random error. The amount of charge the x-ray generates in the detector is vulnerable to random variations and the electronic circuitry inevitably contributes noise to the signal.04 and 8. The breadth of each peak in an x-ray spectrum shows that the energy of an individual xray cannot be measured exactly. In the creation of the spectrum there are errors associated with the assignment of an energy level to any detected x-ray photon. It is also possible to determine from a group of means the standard deviation of the mean which is also given by the formula: σN = σ N We can use this to evaluate how close a single measured value of the mean is to the "true" value of E. The statistics of this process are governed by the Poisson distribution. is the information that we use to determine the elemental composition of the sample. which is not controllable. For example at 10 KV the copper K lines at 8. This energy distribution can be assumed to be an example of a normal (Gaussian) distribution with the breadth of the distribution indicated by its standard deviation (σ).000 and σN is only 1eV.000 individual counts we can take σ as 100 and N as 10. Counting errors affect how well we can measure the rate at which x-ray photons are detected by the detector. a series of energy measurements of x-ray energy E will form a distribution about a mean value. Counting the number of x-ray photons hitting the detector over some time period is critical in determining how much of an element is present in the sample.91 keV are not efficiently excited in contrast to the copper L lines at just below 1 keV. which has a standard deviation determined by: σ= n n is the mean number of x-ray photons detected where 19 . Systematic error is somewhat controllable and can be minimized. This gives us confidence that the mean of the peak is very close to the true energy of the x-ray. If a spectral peak has a standard deviation of 100 eV and is the result of detecting 10. but its magnitude can be estimated from theoretical considerations. Statistical Considerations The x-ray spectrum. In a normal distribution 68 percent of all measurements lie within ± 1σ and 95 percent lie within ± 2σ. which includes instrumental factors such as errors in calibration. Consequently.

Energy Dispersive X-ray Microanalysis For large values of n . If on the other hand our peak had 10. This can be shown by the relative standard deviation: ε= 1 n Thus. the narrower the distribution relative to the mean. the Poisson distribution can be considered Gaussian. if we have a spectral peak representing a single element which has 100 counts then we can say with 95 percent confidence that the counting error is no greater than 20%. so 95% of all measurements of n lie between ± 2σ. Therefore. the larger the value of n.000 counts then we would be 95 % sure our counting error would be only 2%. Clearly it is better to have more counts! 20 .

To setup the microscope for EDS work. By viewing and listening to Volume 3. not the electronic focus knob. Volume 3 is packed with detailed information about the ISIS software. Set the working distance to 15 mm using the large knob on the top of the sample chamber. There is also a 3-volume set of manuals. You can learn about the ISIS system by following the tutorial provided by Oxford. Load the STARTUP =20kV= conditions from the PF5 window on the JEOL 6400. Wait for the vacuum to drop below 10-5 torr. which is a good reference source not designed for cover to cover reading. These conditions should include: CL COARSE – 7 CL FINE .75 OL COARSE – 124 OL FINE – 544 Saturate the beam in the normal way. In addition. Check to see that Aperture 2 is selected. interface electronics mounted in a blue box. Volumes 1 and 2 contain general information about x-ray microanalysis. Focus on the sample using the Z-axis knob. The Link ISIS Operator’s Guide. The Oxford Guide to X-ray Microanalysis. you will need to be able to run the SEM. to start ISIS. Microscope Setup The basic principal of many analytical systems is to stimulate the sample in some way. Many of the steps described in the following paragraphs are designed to ensure that the analytical conditions are identical each time the Oxford EDS is used. and to collect images. which resides on 3 CD’s in the SEM lab. Because there are so many good sources of information. Adjust the stigmators. For important quantitative work. The computer communicates with the detector and with the SEM via the blue box. You can use this time to explore your sample. to measure the response of the sample. this manual will focus on the daily operating details. Load a carbon-coated sample in the microscope. you can learn many of the important features of ISIS. you will need to learn about the ISIS software.Oxford Link ISIS 300 Oxford Link ISIS Introduction The Oxford Link ISIS EDS system consists principally of a liquid-nitrogen-cooled detector crystal mounted in an SEM port. To use this system. If the responses are to be comparable (giving good analyses). and a PC running Windows 98 and Link ISIS software. and to compare the sample’s response with the responses of a set of standards of known composition. and you will need some familiarity with Windows 98. 21 . there are help files included with the ISIS software that can be viewed on screen. wait approximately one hour for the instrument to stabilize. the experimental conditions must be identical and the sample and standards must be similar.

This will open a window like the one at the left (but blank). Clicking on the round Start button on the upper left of this window will cause the ISIS software to acquire the current SEM image (SEI or BEI. click on the blue Labbook button to get the Labbook screen. where the # is an integer assigned to the job when it is created. the Welcome Window will appear. depending on the SEM settings). Now you must choose a job. which will ordinarily be you. This is a digital image (like the right SEM screen) and will improve by averaging over time. 10 keV range). Normal 22 . if you want to change jobs or need to define a new job. as well as the speed of acquisition. Next. To do so. However. it can be added from the Edit menu. you must click on the Jobs button. ISIS will remember what job you were working on the last time. It will be simplest to keep each project as a separate job. you can begin x-ray analysis. If your name is not on the list. SiLi detector. After a message about “Resetting Hardware”. select the Autobeam button. are determined by the Image Setup choices in the Edit menu. Now you must choose an operator. The most important thing about choosing a job is to be sure it uses AC2 (Analytical Configuration – 2. Image Aquisition Once the beam has stabilized (as demonstrated by little change with time of the Faraday Cup readings). Having chosen an operator and a job. The ISIS software will use or create a folder for the job in the AC2 folder with the path C:\ISIS\AC2\J#. 1024 channels. ISIS is ready to start collecting data. The size and detail of the image (# of pixels).Oxford Link ISIS 300 ISIS Startup ISIS can be loaded by selecting “Link ISIS” from the Start menu.

This will open a blank spectrum window. To make a fine adjustment. Beam Current Setup Once you have acquired an image. with peaks for energies corresponding to the x-rays emitted in the 0-10 keV range by the sample. If the current is not -. The image on the left screen of the SEM will blank because the electron beam is now a tiny spot. or exported as a tiff file now using commands in the File menu. saved. If you want a high quality image for printing. As the spectrum appears. The image can be printed. X-ray Spectrum Acquisition To collect the first x-ray spectrum.0025 nA. move the cursor on the right SEM screen to illuminate the FINE CL value. return to the Labbook window (maximize it) and select the X-ray Analysis button.) Now you must make set the beam current. remove the Faraday cup. You should hear a click on the column and see a (negative) current reading on the Keithey Meter. OPTICS window on the right SEM screen. especially for BEI. A graph should appear. The images will be more grainy and the brightness and contrast settings will have to be changed. which changes the COARSE CL setting. Insert the Faraday Cup by pressing the blue PCD button to the right of the SEM screen. However.6000. if you can navigate under these conditions. you will need to adjust it as follows: Press PF2 to get the EOS-1. Make a coarse adjustment using the Probe Current knob on the left JEOL keyboard. You may find it convenient to keep the SEM at a beam current of –. (Note: A right-mouse-click on the image will return control to the SEM. choose.6000 is achieved. such as the one on the next page.6000 ±.Oxford Link ISIS 300 operation will use Fine Resolution (256x192 pixels) and Medium Acquisition Speed. Press the round Start Acquisition button on the upper left of this window to begin counting the x-ray photons. there is less setup for each analysis. You want this current to read -. left-mouse-click at a spot on the image that you wish to analyze. This current gives the best result for the Oxford EDS system and is the current that is used to collect standard data. it is important to check the count rate and deadtime. When a beam current of -. press the square Stop button. You can adjust the brightness and contract of the image using the Contrast Enhancement button. When you are satisfied with the image. High Resolution (1024x768 pixels) and Slow Acquisition Speed.6000 for the rest of your work.) Use the + and – keys on the upper right of the right SEM keyboard to make the FINE CL adjustment. normally to 11. A cross ⊕ will appear marking the spot. 23 . (Use the ESC key to move the cursor from the left SEM screen to the right SEM screen if necessary.

Another common problem is that you did not set the beam current properly. The ISIS software is set to collect data for a preset time of 50 seconds. the Acquisition Status window appears. You can press the square Stop button. the actual analysis will take 80-90 seconds. It is also possible that the X-ray Acquisition Setup (an Edit menu item) was previously set for Optimum Acquisition Rate rather than Best Resolution. You do not have to wait for the full time if you are only doing qualitative work and want to try other points. This is “livetime”. If the readings are different. or all 24 . A common problem is that you forgot to remove the Faraday Cup.Oxford Link ISIS 300 When the Start Acquisition button is pressed. You can label individual peaks. This will open the Identify Peaks window. Identifying the Elements Present You can easily identify most of the common elements in geological samples by first pressing the Identify Peaks “?” button. Check to see that the % Deadtime reading is 30-40% and that the Acquisition Rate is 2. A right-click of the mouse on the peak of interest will bring up a list of possible peaks in the Identify Peaks window. Because 30-40% of the time is deadtime.5 kcps. there is a problem with your setup.02. or simply use the mouse to select a new point on your image (which will restart the preset time clock).

When the elements have been selected. press the Quantitative Analysis “%” button on the X-ray Analysis window to open the SEMQuant Window. which is located in a corner of the thin section and one-inch disk sample holders. and the number of ions in the formula. which will make later data manipulation easier. |Operating conditions must be the same for the 25 . Quantitative Analysis To quantify your analysis. You can also save the results to another file. Be sure the elements you wish to analyze have a green bar over them in the periodic table. Setup for the analysis as described above and acquire a spectrum. An analysis will appear in an SEMQuant Results window that includes Weight % (Element %) and Atomic % of the elements. You can print a copy of the analysis by selecting Print from the File menu. you must analyze the Cobalt Standard. these will ordinarily be Combined element by Stoichiometry. by pressing the Label Peak and Label Series buttons.Oxford Link ISIS 300 the peaks for an element. press the Quantify button on the SEMQuant window. you must periodically (every hour or two) perform a Quant Calibration. select the Nos. of ions Calculation from the Edit menu and enter the appropriate value. Finally. You can also paint a window for an element (for x-ray mapping) using the Paint Window button. Quant Calibration To ensure that you have the best analyses. Choose Copy Text from the Edit menu to paste the analysis into a Microsoft Word document. If you want to see a mineral formula calculated from the analysis. Weight % (Compound %) of the oxides. choose the Processing Options. To do so. Select the elements present in your sample to analyze using the periodic table that appears when you press the Select Elements button. For many elements you can choose the standard to be used by pressing the Configure button. Choose Copy Table from the edit menu to paste the final table into an Excel document. For minerals. The standards used are also listed. with Oxygen as the combined element.

Press the Calibrate button. Advanced Features The Oxford ISIS system has a number of other features that may be of use.6000 nA beam current. Acquisition Setup must be changed to Optimum Acquisition Rate using the Edit menu of the X-ray analysis window. select Quant Calibration from the Options menu of the X-ray Analysis window.Oxford Link ISIS 300 Cobalt as for your unknowns. Note the deadtime will be higher (40-50%) for the Cobalt than for most minerals (30-40%) for the –. Maps can take minutes to hours to collect.xls. This feature will allow the user to collect x-ray intensity maps for specified elements. The Gain Calibration window will open. One that many geologists will use is x-ray mapping. Use the manuals to learn more about mapping and other advanced features. When the spectrum is acquired. The Quant Calibration data are saved with each spectrum so that data collected under different conditions can be compared. C:\Oxford Instruction Files\Quant Calibration Record. 26 . Windows for the elements of interest must be painted using the button in the Identify Peaks window. depending on the resolution required. To collect an x-ray map. Enter the new calibration values in the Excel spreadsheet. The Mapping button is then selected in the X-ray Analysis window. A test spectrum must be acquired.

Section 3 Appendices 27 .

28 . and remove your specimens to a safe storage container.Sputter Coater Appendix A SPUTTER COATER 1. Replace the glass cylinder and close the lid. Be sure the high voltage control switch is OFF and the high voltage dial selector is at 0. turn high voltage switch OFF. After coating. not the raised ring around the periphery. Press ON the high voltage switch c. turn the needle valve toward increase d. Check that the vacuum seals are clean and greased and reassemble the machine. dial to 0. then ADJUST the current so that the current meter reads 10 milliamps. Do not pump it down. 5. Lift the lid off the vacuum chamber. You are now ready to begin coating: a. Do not overtighten this needle valve. TURN ON the main power switch. turn the needle valve toward decrease >> if the current is too low. CLOSE the vent toggle valve (down) and CLOSE the increase/decrease needle valve (turn to full decrease). Allow about 10 seconds for the instrument to stabilize and the vacuum chamber to outgas. set high voltage dial to 9 b. Wait until the vacuum reaches 45 millitorr on the vacuum gauge. Open the lid. you may need to touch up the current flow occasionally to keep the meter at 10 milliamps. This prevents messing up the vacuum grease on the top and bottom rims. CLEAN THE CYLINDER inside with a paper towel wet with absolute EtOH or acetone. High voltage switch should be OFF. main power OFF. 3. 2. and immediately OPEN the air inlet toggle valve (lift). leave it under atmospheric pressure. This necessitates a considerable number of turns of the valve stem. Place your specimens on the central area of the pedestal. Then OPEN (turn toward increase) the increase/decrease needle valve to bring the pressure up to (and stabilized at) 50 millitorr. >> if the current is too high. Carefully remove the glass cylinder and lay it on its side where it won't roll off the counter. 4. This will take anywhere from a few minutes to perhaps fifteen. Coat your specimens for 2 to 3 minutes. remove the glass cylinder (lay it on its side where it won't roll).

The final lens contributes very little to the demagnification.hence demagnification. it can also be reduced on the viewing screen by slowing down the scan speed. This setting can be changed by turning the probe current knob. it may or may not be the proper setting for further specimen examination. as mentioned above. This can happen if one increases the focal length of the condenser lenses by turning the probe current knob counterclockwise. but the smaller spot size results in a need for more amplification of the signal and thus noise (or graininess) is introduced into the image. This graininess does not show up on the pictures. In theory. The beam spot is made smaller and the resolution of the image is increased.e. one must increase the signal level. More electronic amplification (contrast setting) is needed to view the image. The other option to increasing the signal output without increasing the probe diameter is to slow down the scan speed so that the probe spends more time in each position in its raster. it will start up with the magnification set at 100x and the condenser setting at 7. the condenser lens current in increases and the focal length of the lenses is decreased. the smaller to spot size the greater the resolution and the sharper the image. The SEM has many operating parameters which can be adjusted over wide ranges to accommodate a large variety of samples. The message is that you want a smaller spot size at the higher magnifications to give you the resolution you may need. as outlined in the next section. When you turn on the instrument. and the noise level or graininess increases.due to smaller spot size and fewer primary electrons. and a smaller percentage of the electrons in the beam pass through the aperture below the condenser lenses. images will not be sharp enough to be useful. the noise level becomes so large that the signal can no longer be recognized. Resolution too is linked to magnification. which determines the probe or beam diameter or spot size. the beam diverges more after it leaves the stronger lenses. for resolution is dependent to a large degree on the area covered by the beam or probe as it is focussed on the specimen by the final (objective) lens. the signal to noise ratio could become so small that the image is too noisy to be useful. for the scanning electron microscope depends on the degree of resolution desired in the image. a smaller spot size necessitates increased amplification of the signal and this results in increased noise or graininess of the image.the image formed of the object is smaller than the object itself . 29 . If carried too far. Then not as much amplification is necessary and there is a reduction in the noise level. The signal level can be increased if a larger percentage of the electrons leaving the gun were to reach the specimen. The optimum condenser setting must be determined by the actual operating conditions in use at the time. To obtain a large signal to noise ratio for easy viewing of the image. If carried too far. this is accomplished with the probe current knob. this is a medium spot size. The proper condenser lens setting. The final spot size can be adjusted by changing the strength of the condenser lenses. This means there are fewer primary electrons forming the beam and thus there will be fewer secondary electrons generated at the specimen. However. you need a feeling for what happens in the electron-optical system when you change it.Condenser Lens Setting Appendix B CONDENSER LENS SETTING (PROBE DIAMETER) This description is taken with some modification from an old AMR publication. But this allows the spot to become larger and reduces the resolution. chosen as a middle point to initially obtain a usable image. thereby reducing the beam divergence beyond the aperture angle. i. This instrument uses two condenser lenses to demagnify the relatively large beam source that is formed in the electron gun. When one increases the probe current setting by turning the knob clockwise. This is sort of like looking through the wrong end of a telescope . However.. The signal is reduced . In order to know when it is appropriate to change the spot size. There is also a final lens to focus this demagnified or reduced beam onto the surface of the specimen.

Thus resolution is increased. this decreases the number of primary electrons reaching the specimen. Remember that the spot size is bigger so the resolution will suffered a bit. When you have found some particular feature. When you turn down the magnification. This increases the focal length of the condenser lenses. low noise image. 30 . So you decrease the condenser lens setting by one or two (or more) positions. and touch up the contrast and brightness. You can try now to reduce the scan speed. after some fiddling with contrast. you may want to go to a lower magnification to orient yourself and/or perhaps search for some particular feature. As outlined above. that is. The spot size resulting from the 7 setting of the probe current may not be big enough (have enough electrons in it) to yield a signal to noise ration suitable for clear. the image may look grainy. thus increasing the signal. reduces beam divergence. as well as reduces the spot diameter. so you increase the magnification. The result. this is not noticeable at lower magnifications because the resolution if the viewing screen and your eye become the limiting factors. brightness and focus. increases the diameter of the spot. and increases the percentage of the beam that reaches the specimen. but the image is still not sharp. you may want to examine it at a higher magnification. is a clear. Then you must increase the probe current or strength of the condenser lenses.Condenser Lens Setting When might you want to change the condenser settings. low noise viewing at this rapid scan speed. focus and correct the astigmatism. However. move away from the initial setting of 7? Once you have obtained an initial image. as is the noise.

the resolution of the scanning electron microscope.000X 20 nm. probe if you can’t visualize the extra resolution because you haven’t magnified the image sufficiently. in order for a final image to appear with maximum sharpness to the eye at 100.2 mm.e.2 mm. the true size of a 0.000X 100 nm. etc.. to maximize depth of field. is determined by the diameter of the electron beam (probe) as it is focussed on the surface of the specimen. This is easily accomplished by using the condenser lens current knob.000X 40 nm. This means that any magnification above around 50. 5.000 is truly “empty magnification”.000X is pretty much the upper limit of useful magnification. operated in secondary electron mode. it is not necessary to pursue those very small beam diameters.570X 7 nm. one must select the appropriate beam size to permit maximum sharpness and detail (i. This is an unattainable goal. so 20. working at the typical distance of 39 mm. As implied in the preceding section. 50. so as to provide optimum viewing conditions for specific magnifications. 500X 400 nm. 31 . 20. 10. Magnification Appendix C Resolution vs. feature found in the image. IMAGE FEATURE SIZE vs. it must be enlarged to at least this size. match the probe diameter and the magnification. one finds the actual size of the smallest feature capable of being resolved by the eye (0. with all the attending problems of decreased signal. For example. or better. either on the viewing CRT or in the micrograph.000X 4 nm. resolution) in the image.000X!! Much of our work is done at magnifications less than 5. In other words. one typically varies the beam diameter as one changes the magnification. etc. increased amplification and accompanying noise. it merely makes small and fuzzy portions of the image appear larger and fuzzier. 2. At these lower magnifications. and at 8 mm. 200X 1000 nm. with a real good specimen. turn it down for a larger spot. Magnification In the final analysis.) ** instrument resolution This table contains some rather interesting information..000X 2 nm. the best attainable resolution is 10 nm. In other words. (1 µm.2 mm) at a number of different magnifications. the instrument and its camera. the optimum resolution attainable is about 3. 28. Don’t use a 10 nm. must be resolving at 2 nm.000X magnification.. making the spot smaller. in other words. This means that in order for the detail in the specimen to be appreciated clearly. One might wonder why they built this instrument with a maximum magnification of 300. Turn up the current to further demagnify the probe. working distance. AMR publication. and this optimum beam diameter varies with the magnification. with the instrument working at its best. In the table below. For the average user. The magnification refers to the final magnification of the micrograph and includes any enlargement done during printing. FINAL MAGNIFICATION Real size of 0.5 nm.Resolution vs.000x.2mm Magnification feature in image** 100.000X 10 nm. for on a very good day. One needs to keep in mind that the average human eye can has the ability for spatial resolution on the order of 0. if the eye cannot see anything smaller in specimen dimensions than 40 nm. This description is taken with some modification from an old.

This helps stabilize the lipids and adds a potential source of secondary electrons to the specimen. flies). Their preparative techniques may be equally diverse. Those which may not be dry. seeds. etc. Those samples which are soft and wet. it eliminates water from the specimen. The methods of applying the fixative are varied as well. etc. pollen grains. This group might include some hard bodied insects (beetles. and it also may be advantageous because it hardens the tissue so that it can be cut to expose fresh. usually either ethanol or acetone. thus preserving the morphology in a life-like state. and OsO4 is most frequently used. into the circulatory system. Fixation This applies especially to those samples represented in group c. bone. dehydration is an essential process. above. This will stabilize the tissue. but are fairly rigid and resistant to distortions which might result from desiccation. wood. Perfusion fixation must be used. one fixes in a buffered aldehyde solution. before they are air dried and helps to reduce shrinkage and minimize collapse and distortion. for the interior may be poorly fixed. c. hair. under gentle pressure. In this latter regard. such as those from group b. Those materials which are already hard and dry. These need to be dried carefully so as to avoid distortion. typically by slowly replacing it with an organic solvent with a low surface tension. Drying Air drying is useful for those specimens that can stand it. This section intends only to outline on very general terms some of the procedures more commonly used for viewing specimens in the secondary electron mode. immersion fixation may well be unsatisfactory. They are likewise in need of chemical fixation in order to preserve structure. metal. where it provides a solvent more amenable to exchange with the critical point drying solvent (liquid CO2). such as mineral specimens. Dehydration For SEM. helping it to resist collapse during drying. Often simple immersion fixation will work. and then more OsO4 in an effort to get more metal into the sample. The specimens are simply dumped out on a piece of lint free paper and the solvent is allowed to evaporate. The disadvantage is that perfusion is more difficult and time consuming. for example 2% glutaraldehyde in 0. 32 . Fixation stabilizes and cross-links the proteins and to some degree the lipids of the specimen. internal surfaces and structures. This process would apply to those specimens in groups b. this includes most biological specimens. and making the specimen more resistant to subsequent treatments. pH 7. investigators may follow postfixation with an incubation in thiocarbohydrazide. Type of specimen a.. hopefully. b.4.1 M cacodylate buffer. A postfixation is also desirable. Most commonly.Specimen Preparation Appendix D SPECIMEN PREPARATION A wide variety of specimens may be viewed with the scanning electron microscope. here the fixative is injected. It is also done prior to critical point drying of those specimens in group c. scales. If one wants to visualize the internal structures. especially if the sample is small or if only exposed surfaces are going to be examined.

Then the pressure is slowly reduced while the temperature is maintained.Specimen Preparation A more elaborate method must be used with more delicate specimens. the chamber is sealed and the temperature.) are coated with a thin layer of gold and palladium before viewing. they are deflected around inside the chamber. The sample is left behind.. allowing all the CO2 to escape the chamber as a gas. There are a number of solvents which have extremely low surface tensions. Because of this directionality. and thus the pressure too. completely dry. Coating This step is not necessarily essential. the specimen must be rotated to try to get an even coat. The metal molecules leave the source and move in essentially straight lines until they strike any surface. Coating can be done in a vacuum evaporator where the metal is evaporated under a high vacuum. The water in the tissue is replaced by acetone or ethanol. This surface layer provides an ample supply of secondary electrons for the signal. See Appendix A for operational instructions. since they are in a poor vacuum. The procedure is designed to remove the solvent from the specimen without damage from the surface tension forces which would cause it to collapse as the fluid interface recedes through the specimen. they serve to coat all the surfaces evenly. and this in turn is replaced by liquid CO2 under pressure and at 0°. They too condense onto the surface of the specimen. and following the exchange. but typically specimens (from groups a. It is known as critical point drying and is applied to cells and tissues and other specimens that are likely to undergo distortion and shrinkage as a result of drying. The procedure uses a specifically designed machine. is elevated above the critical point for CO2. forming a film. This takes place in a special chamber. 33 . b. and c. A better procedure uses a sputter coater. and indeed for some work it may be undesirable. Here the metal molecules are physically removed from the target source by ion bombardment. but since they lack directionality and are moving in random directions. Peldri II® and hexamethyldisilazane are two examples. Then they condense out onto the surface. and the operational details can be found in the instruction manual in the lab. These too are now finding their way into the specimen preparation procedures. including the specimen..

and the CHAMBER VENT VALVE is CLOSED. Turn Main Power Switch ON. if they differ very much from those suggested in these instructions.Vacuum Evaporator Appendix E VACUUM EVAPORATOR Start-Up Procedure NOTE: Observe the pump-down times. 2. 34 . or John Brady. the MAIN VALVE is CLOSED. After about 3 minutes or less. the reading on the Pirani gauge should be less than 2x10-2 mbar. The valve is located in the sink in adjoining room. 1. Turn the Rotary Pump ON. 4. Turn ON cooling water to diffusion pump and check for adequate flow rate. 3. Dick Briggs. This switch is located on the right side of the instrument (see above diagram). Check to be sure that the Backing-Roughing Valve is in the MIDPOSITION. notify Judith Wopereis.

Vacuum Evaporator 5. Turn Diffusion Pump ON. Unscrew the power cable and remove the electrode assembly by first loosening the set screw at the top of the assembly 35 . During this time one can vent the bell jar.place it bottom up in the box provided! 5. They should be lined up directly under the slot in the electrode assembly. Do not allow to roll onto the floor . 2. 6. If you are continuing. follow the pump-down procedures on the next page . you will need to insert a new electrode. 3. Electrode Replacement (to be performed after each firing) If you are finished using the insturment. after the 20 minutes. OPEN the CHAMBER VENT VALVE. then reassemble the electrode holder without a new. insert specimens and set up electrodes (see following section for instructions on this). or. starting with step 4. Turn the Backing-Roughing Valve to BACKING. sharpened electode. proceed through step 3. Check that the Penning Gauge is OFF. Wait 20 minutes to allow the diffusion pump to warmup. be sure to keep gasket clean. if not already closed. Sample Insertion or Change Procedure 1. Samples should be placed on white paper on the support platform to help you see the thickness of the coat and to help keep the sample holder platform clean. If you are just starting up. follow through step 5. CLOSE the MAIN VALVE. 1. Remove cage and Bell Jar. 4.

Loosen set screw B and remove the flat-ended electrode. notify Judith Wopereis or Dick Briggs. 3. Turn Backing-Roughing Valve to ROUGHING 3. 4. 2. CLOSE Chamber Vent and replace Bell Jar and Cage. IT IS ABSOLUTELY CRITICAL THAT THERE IS A GAP BETWEEN THE METAL COVERS HOLDING THE TWO ELECTRODES. Pump-Down Procedure NOTE: Observe the pump-down times. Turn Electrode Selector to position 1. OPEN the Main Valve. use fine sand paper to smooth the flat surface and then replace in the holder. Turn the Evaporation Control Toggle Switch to PULSE. Use position 7 for a “thick” electrode 36 . Turn Penning Gauge ON when vacuum reaches 6x10-2 mbar. Wait until vacuum reaches less than (to the left of) 1x10-4 mbar on the Penning gauge. Turn Electrode Power ON. if they vary very much from those suggested in these instructions. sharpened electrode. 2. This spring tension will hold the two electrodes together as the tip of the sharpened electrode is burned off. If necessary. 5. 5. replace the sharpened electrode with one of the appropriate size. When Pirani Gauge reads less than (to the right of) 1x10-1 mbar (less than 1 minute) turn Backing-Roughing Valve to BACKING. 1. if not already done. The instrument should reach 10-4 mbar within 5 minutes. 4. If you are going to immediately use the coater. 4. Turn Electrode Voltage to 8. IF THESE TWO PARTS ARE TOUCHING THERE IS SHORT CIRCUIT WHICH CAN SEVERELY DAMAGE THE INSTRUMENT (see above figure). Coating 1. remove the used. 3. Then pull back the end assembly C to apply spring tension to the sharpened carbon rod and tighten set screw A. maximum. Loosen set screw A. 5.Vacuum Evaporator 2. adjust the set screws on each cover.

Turn Diffusion Pump OFF. CLOSE the Main Valve. PULL THE PLUG AND RUN!! LET SOMEONE KNOW WHAT HAPPENED (notify Judith Wopereis. If more samples are to be coated.Vacuum Evaporator 6. The system must be completely pumped-down and left under high vacuum (Follow Pump-Down Procedure). Turn main power switch OFF. Apply coat by pressing the PULSE button in approximately one second intervals. Turn cooling water OFF. 5. it should come down to less than 1x10-4 mbar. Check Penning between pulses. Prior to shut-down. IF THERE IS TIME. Turn Rotary Pump OFF. TURN OFF THE PENNING GAUGE. 4. CLOSE THE MAIN VALVE. insert new samples during this time. Approximately six pulses will generally apply a good coat. On completion of coating: turn Electrode Voltage to 0: Turn Evaporation Control Toggle Switch to OFF: Turn Electrode Power OFF: Turn Penning Gauge OFF. Watch that the current does not reach 100. Shut-Down 1. Leave cooling water on for at least 20 minutes and continue to back the Diffusion Pump (Backing-Roughing Valve in BACKING position). EMERGENCIES: IN CASE OF FIRE. Dick Briggs. or Bob Newton). the Bell Jar and Sample Holding Assembly should be thoroughly cleaned and a new electrode left in the Electrode Assembly but with no spring tension applied. Log out in Log-Book. IN CASE OF A REAL DISASTER. 6. Follow Sample Insertion or Change procedures (above) to retrieve samples. turn Backing-Roughing Valve to MID position. AND TURN OFF THE DIFFUSION PUMP. 37 . 2. John Brady. pull the plug as well. After 20 minutes or more. OR POWER FAILURE. 7. turn OFF the Penning Gauge. 3. then follow instructions for Electrode Replacement and Pump Down. FIRE DRILL. Once a high vacuum has been achieved (less than 1x10-4 mbar). 7.

and select the Crop Tool. digital imaging in the microscope lab is here to stay. b. then use Image.Image Manipulation Using Photoshop Appendix F IMAGE MANIPULATIONS AND LABELLING USING PHOTOSHOP by Tina (Weatherby) Carvalho Biological EM Facility. In 4. such as one obtained with NIH Image. for reports. c. Start up AdobePhotoshop 3. and the ethics of using it to manipulate visual data is being hotly debated. d. overhead transparencies. use the Crop Tool. but that is the subject for another article! The learning curve for Photoshop is steep. If your file doesn't have an extension recognizable to Photoshop. 2.0 you must Select All of the image.0 press C to select it or hold the mouse button down over the rectangle selection tool in the Toolbox to get a pop-up menu of other selection tools. Photoshop is extremely powerful. and am beginning to find ways to assist the clients in our multi-user facility with their digital imaging needs. Generally. I strongly feel that any manipulation beyond what is outlined here would be unacceptable. . then find and select the filename for an existing image. In 3. To this end. University of Hawaii Text scanned from Microscopy Today. Crop the image if necessary. and from a flatbed scanner. and the majority of the available books deal at length with color management and effects. however are more interested in faithful reproduction and presentation of our visual data. a. Adobe PhotoShop is the software of choice for image adjustment.0. The following is a step-by-step mini-tutorial designed to lead you through the rudiments of Photoshop. then double-click inside it (4. or contributions. Image Adjustment 1. comments. We can acquire digital images from our FESEM. 4. these images need to be manipulated for use. If you have any corrections. please feel free to e-mail me at tina@pbrc. selecting a slightly smaller size than your image.0 the Crop Tool may also be resized and rotated before cropping. Drag the crop marquee around the portion of the image you want to keep.0 or 4. and publication.edu. #97-10.0 you may discard a few pixels around an image by selecting ImageCanvas Size. In 3. We. 3. While I am one of those fogies who insist that digital images are nowhere near as good as photographic prints. For more cropping. as scientists. In 4. December. In 3. 1997 Ready or not.0) or click once with the scissors cursor (3. Select File-Acquire to obtain an image with a scanner or other plug-in. accounting for more than 80% of the digital image-editing software market. I've tried to distill the contents of many books and a two-day workshop down to the basic techniques of interest to microscopists. just as one would manipulate a photographic print to correct any deficiencies in exposure or area of view. Select File-Open. taken from a manuscript I am preparing.0) to crop. slides. tentatively titled 'Photoshop Distilled: Basic Black and White Image Adjustment and Layout for Scientists'. I've also bowed to the inevitable. or. and I'm sure there are some who will feel that even this is too much.0 click on the Crop Tool in the toolbox. from our light microscope with a CCD.hawaii. 38 . Clients use their images for image analysis. click on Show All Files to find and then rename it. transport to others scientists via the Internet or World Wide Web. and then placing your image in the center or to one side.0 or 4.

meaning that some information is lost.g. Select Image-Adjust-Levels and get the gray levels histogram b. b. 1. 2) Select a radius of perhaps 1 to 4 pixels. Resampling or resizing up (not a good idea) or down changes the apparent sharpness. and re-do gamma correction again. format and location for it. will this change your data? a.g. 3) Select a threshold of perhaps 0 to 16 levels... b. or enter a value in the left most Input Levels option box (e. Save your file using Save As.8). move the middle. or enter value in the rightmost option box (e.. 235). white arrow to the left to select the lightest pixels. Check image..0 by selecting View-Print Size). c. 6 Determine if image needs sharpening. 144%). JPEG is a lossy compression scheme. 3) For midtone. select TIFFwith no compression. 4) Click OK. For greatest fidelity and transferability between computer platforms. black arrow under the histogram to the right to select the darkest input pixels.20).Image Manipulation Using Photoshop 5. Best sharpening is accomplished by Filter-Unsharp Mask: 1) Set a value in the dialog box (e. TIFF with LZW compression will compress the file size somewhat with no loss of information. Values less than about 25 will be subtle and more than about 300 will be too dramatic. c. View image in the size it will be printed (in 4. a. The best compression is performed by selecting JPEG and then the highest level of 10 (4. 39 . and select a name. but may not be accepted by some computers. or gamma correction. if needed.g.0). or enter a value in the middle option box (e. but often this loss is unnoticeable. Check your ethics barometer. Be aware that in order to get a good print you will need to adjust your on-screen image until it shows the full range of grays but overall looks too light! This is due to the fact that the image you see on your monitor has already had gamma values applied to it that will not be sent to your printer. Adjust input levels: 1) Move the left..0) or maximum setting (3. 7.g. 2) Move the right.. gray arrow to the left. Adjust image tones: a. Click on OK.

Select the color you want your bar as your foreground color. White can be brought to the foreground by clicking on the curved arrow pointing between the two.g. black will be the foreground color. perhaps more effectively. use Edit-Undo before you do anything else. Size.0 so that you can more readily see the text. Check the anti-aliasing box in the Text options palette if you wish to have PhotoShop soften the edges of the type. a. but note that it still remains a selection until you click elsewhere or otherwise deselect it. Select Anti-aliased. With your image file open. Use the Line Tool cursor lines at the top and side rulers to position and measure the length of the line that you draw by pressing the mouse button and dragging on your image. Make sure you remember what this magnification bar represents. However. Other labeling with text and arrows can be done either with Photoshop (the subject of the next section) or. at high resolutions the jaggedness is not so apparent. b. Type your label in the box. 4. 1.0) or as its own layer (4. PageMaker. Style(s) and Alignment.0). select Select-Hide Edges. such as reduction in print or as a slide. 1. Type applied to an image will have the same resolution as that image. 4 pixels) and make sure that Arrowheads Start or End are not selected. Select Font. Labeling images in Photoshop Addition of type to digital images is best done with software designed for that purpose. Quark Express. To apply text to an image. 5. Position this cursor over the image approximatelywhere you would like the type and click. 2. Normal.. You will get a dialog box. then click OK. and 100% opacity. You may set the units to centimeters. or picas in File-Preferences -Units. click on the tiny black and white rectangles at the bottom of the Toolbox. etc. Enter a line width (e. 40 . 6. If you made a mistake or don't like your line. You mayappend notes to the image by selecting File-File Info. 3. To turn of the marching ants in 3. inches. c. click on the Type Tool (T) in the Toolbox. To get true black or white. such as QuarkExpress. with additional programs such as PowerPoint. no matter what its ultimate fate.Image Manipulation Using Photoshop Applying Magnification Bars Applying a magnification bar in Photoshop makes sense because then it is always available with your image. type is treated as another bitmapped image and. go to Window-Show Rulers. Double-click on the Line Tool to get the options palette. Adobe PageMaker. This information will be available to you each time you open the image. or Microsoft PowerPoint. will exhibit pixelation at low resolutions. Your text will appear in the foreground color and as a selection with marching ants (3. as such. Yourcursor will become an I-beam. With Photoshop.

0) or thrown in the trash in the layers palette (3. Anti-aliasing.g. such as arrows. you can Edit-Undo if it is the last thing you did.0) or throw away the layer (4. In 3. b. and it will move the entire layer. text can be moved with the Move Tool or with the keyboard arrows. The arrow will be in your foreground color. Trust me. e.0).0 text always appears as a new layer. or end where you stop your cursor. In 4. the type. and 100% Opacity. Your best bet is to place arrows in their own layer.0). 4.Image Manipulation Using Photoshop d.0) or as a floating layer (3.. 400%). 4).0. e. In 3. Select Line Width in pixels (e.. cannot be applied to text layers. While selected or in its own layer. Select Arrowheads Start or End. or to the rightpointing black triangle on the top right and select New Layer from the pop-up menu. a. j. allowing you to better position the arrow. I.0 you must use the Move Tool.0 you may hide the marching ants but maintain the selection by going to Select-Hide Edges. depending on whether you want the arrowhead to start where you begin to drag your cursor.g.g.0. In 4. g. go to Edit-Undo while it's still the last thing you did (3. 16%).0 you may move the arrow around with the Move Tool or keyboard arrows.0. double-click on the Line Tool in the Toolbox to see the Line Tool Options palette.0 you can delete this layer now or later. so check it now. d. Non-text additions. is in its own layer (4. Select Shape-Width (e. Save As a new filename so that the labeled image stays separate from your unlabeled one. while still selected. If you make a mistake. In 4. you may want your unlabeled image back someday! 41 . If you don't like your arrow.. Length (e. This also works for 3. To apply arrows to an image.. Normal. f. c.0 and 4. the amounts of which are related to the Line Width. and Concavity (e. 600%).g. Go to Window-Show Layers palette and click on the little folded paper icon at the bottom.0) and can still be deleted with the delete key (3. If this is a mistake. 2. Deselect to paste the label down in 3. f. Click on OK. Drag the mouse across your image to make an arrow. h.

0. For 300 dpi images at about 4" x 5". Add a black line at an appropriate length and width and any other text. A Marquee should be around each object. 2. If you want to save this as image in another format such as TIF or BMP. John Minter email: minter@kodak.0 TO LABEL SCANNING ELECTRON MICROGRAPHS by John Minter. In version 4. go to the pallette region of the toolbar and exchange foreground (usually black) and background (usually white) colors. type the text. Before you de-select the text. Don't merge them with the background layer! 3. 5. symbols. Then go to the "Edit" menu and select "Stroke" chose a 2-3 pixel stroke width "outside" the letter. you preserve the original micrograph in the background layer. We use Adobe Photoshop. By using the layer. You have to move the selected region up then down with an arrow key.Using Photoshop 4. text. then you have to Merge the layers and save the image in that mode. (This is done in the PC with the Ctrl-shift-arrow key in the PC). Deselect (ctrl-D) 8. be sure to select the outlined text tool so that the letters are surrounded by marquees when you are done typing. This makes an outlined text in any font on your system. In that layer in the font and font size that you want. You can make them look like real Rub-ons with another technique and offsetting the white a little. (You are going to write a white border around each Marquee.0 to Label Scanning Electron Micropgraphs Appendix G USING PHOTOSHOP 4. What this does is to select all of the objects in the layer individually.) 6. This will write a white border 4 pixels wide around all of the selected black features. Select all (ctrl-A in the PC) The marquee will be around the whole layer. that you want to put on the micrograph. I suggest a font size of about 14 (Arial) with a Width of about 3-4 pixels for the stroke width. from the Microscopy Listserver This is the recipe that I use in Photoshop 4.com 42 . Select the foreground color as white. 7.0 to put Black on White scale markers. etc. you can use the info window to draw lines to particular lengths. but that is another story. 4. Go to Edit-Stroke and select the width of the white line you want (Width) and select the Outside option. Note: you should have anti-aliasing selected for all this. merge those layers. and symbols onto micrographs. If other layers are created when new text is added. arrows. Type your text on the image. 1. This technique works very well for me. Create a layer (Photoshop 4. You must use Photoshop image mode for the layer option.0 does this automatically with text tool). The results look just like the rub-on transfers that I used to use.

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