OPERATION OF THE HITACHI S-450 SCANNING ELECTRON MICROSCOPE by Doug Bray Department of Biological Sciences University of Lethbridge Revised September, 2000 Note: The terms in bold in this document represent labels on the instrument controls. A. Turning On and Standby 1. Turn on the water supply by flipping the first (right hand side) red handle to a vertical position. 2. Turn on the scope by flipping the EVAC POWER switch on the front of the console to ON. 3. Wait until the HIGH VAC light on the gun airlock valve (right side of column at top) is green (approximately 20 min). The instrument is now on STANDBY. If there was a previous user, they may have left the instrument in the STANDBY condition which implies the following: - specimen chamber empty - green HIGH VAC light on gun airlock valve on - red HIGH lamp above VACUUM meter on front of column unit on - DISPLAY POWER switch on front of column unit turned off - EVAC button on front of column unit pressed in - TILT control at 0 angle - X and Y controls at centre position (i.e. 20 mm position on shaft) Note: Because of the LaB 6 filament and the Ion Getter pump, it is imperative that you use caution when opening the specimen chamber. B. Inserting the First Specimen 1. Make sure the gun airlock valve is closed, then, push the AIR button on the front of the column unit in, then wait approximately 30 sec for the chamber to fill with air. While you are waiting check to make sure that the Z-control knob is at the EX position. Page1
2. Grasp the specimen chamber assembly by the sides and pull it out far enough to allow stub exchange. DO NOT HOLD ONTO ANY OF THE SPECIMEN MANIPULATION CONTROLS AND PULL SLOWLY. 3. Use the forceps provided and insert your specimen stub into the hole at the centre of the stage. Use the screwdriver to secure the stub in place. 4. Gently push the stage assembly back in place. 5. Push the EVAC. button on the front of the column unit in while holding the stage assembly in place to ensure a good vacuum seal. 6. Wait until both the HIGH vacuum light on the front of the column and the HIGH VAC light on the gun airlock valve turn green (about 5 min.). 7. Open the gun airlock valve by turning it clockwise about 90, pulling it out till it stops, and then rotating it counterclockwise about 90 again to lock it in the open position. Note: If you hear a buzzer during this operation, close the valve immediately and call me. C. Obtaining an Image 1. Flip on the DISPLAY POWER switch on the front of the column unit. 2. Select the accelerating voltage required by turning the ACC. VOLTAGE (kv) to 2, 3, 4, 5, 10, 15, 20, 25 or 30 kv. Settings of choice are normally 5 to 15 kv. 3. Push the H.V. (KV) ON switch (red light under the selected kv should come on. 4. Obtain a beam by slowly (how slow depends on when the instrument was last used) turning the Filament control up until it reaches the correct value as determined by maximum height of the waveform (you'll need personal instruction for use of the waveform). Next, turn up the Bias control until it just begins to reduce the height of the waveform. You will have to perform these adjustments periodically while using the instrument. Below is a chart of approximate values for different kv's. Acc. Volt. Bias Filament 4 kv 35 450 5 kv 40 500 10 kv 80 435 15 kv 80 450 20 kv 100 440 25 kv 150 465 Page2
At this point there should be several green lights lit up along the color bar indicator of the EMISSION CURRENT meter. If the scanning mode selector switch has been left on the first slow scan position (three arrows), as it should have been, you can change it to full frame rapid scan. This will allow you to optimize the image using the following controls: MAGNIFICATION : Set this to the lowest value while obtaining image, and then change to whatever magnification is appropriate. GUN ALIGNMENT : With the display mode waveform switch pushed in, adjust the X and Y knobs to obtain the highest waveform on the screen. WORKING DISTANCE : This should be adjusted to the correct position with respect to the specimen height. (#1 = 5 mm; #2 = 10 mm; #3 = 15 mm; #4 = 25 mm; #5 = 35 mm, values are approximate) BRIGHTNESS : You can either use this manually (AUTO switch on the BRIGHTNESS color bar indicator in out position); or in automatic mode (AUTO switch in). CONTRAST : Use this control to adjust the degree of contrast in your image, a very important factor. Normal setting should show red lights on the left, and one or two green lights in the middle. COND LENS : Because of the LaB6 filament, this control should only be varied between 300 and 450 for normal work. Most useful setting is about 400. 4. Once you have optimized the above parameters, you can work with your specimen in the same manner as you did with the S-500, as most of the same controls are found on both microscopes. NOTE : Unless you are actively using the scope display screen, you should leave the mode selector switch in one of the slow scan positions, usually the first one (three triangles). This will avoid overheating the scan circuitry and prevent costly repairs. D. Basic Operation 1. Specimen Movement The mechanical controls on the stage assembly permit specimen movement in X, Y and Z directions as well as tilt and rotation. If your specimen is higher than normal (> 5 mm) do not raise it beyond the 10 mm mark on the Z axis scale to avoid damage to both the specimen and the lens. 2. Magnification Selection Page3
Image magnification can be varied using a combination of step increments (large silver dial) for large mag. changes, and the zoom control (inner silver dial) for small mag. changes. There is also a low magnification button above the mag. selector switch. 3. Focusing Focusing the image is accomplished using both the mechanical Z axis control on the column desk, and the electronic focus dials on the display console. Start out at low mag. and match the height setting on the Z axis dial with the setting on the WORKING DISTANCE focus dial (i.e. specimen height of 15 mm on the Z axis should roughly correspond to a setting of 15 on the WORKING DISTANCE dial). The COARSE and FINE focus knobs are then adjusted consecutively to obtain the sharpest image. The FINE focus is normally only used for high magnification work (above X 5,000). When focusing critically for photography, it is best to increase image resolution by pushing the REDUCE AREA button on the SCANNING SPEED controls, and increasing the magnification by 2 to 3 increments using the outer mag. dial. While doing this, optimize BRIGHTNESS and CONTRAST so that the clearest image is obtained. Both FINE and COARSE focusing can then be done before returning the image to its original magnification and size. 4. Brightness and Contrast Control The setting of the brightness control can be either manual or automatic (preferred). Set the contrast control by discretion based on your experience with the S-500 SEM. 5. Scanning Mode Switches The first mode button should be pushed in for normal viewing of the image. If you are not actively working with the image on the crt, push the button with the three triangles on it to avoid overheating the circuitry. The third (one triangle) button can be used to optimize the PCI image on the computer screen. E. Changing Specimens 1. Reduce magnification to a low setting (around X 50). 2. Turn the filament off by rotating the dial counterclockwise. 3. Push the OFF button on the H.V. (KV) panel. 4. Centre specimen stage to about 20 mm for each of the X and Y stage controls. 5. Bring TILT control to 0 angle. Page4
6. Rotate the gun airlock valve handle 90 clockwise until it stops automatically, let the vacuum pull it in, then turn it 90 counterclockwise to the lock position. 6. Push the AIR button on the column bench and wait until the chamber is pressurized (about 30 sec.). 7. Pull the specimen assembly out as before. 8. Release and remove the specimen stub and insert another. 9. Close the specimen chamber and pump out as before (i.e. push in to obtain snug seal, hold in place, and then press the EVAC button). Wait approximately 5 min. until the green gun airlock valve light comes on. 10. Push the HV ON button and saturate the filament as before. F. Optimizing Image Quality 1. Accelerating Voltage This control influences image resolution by altering the secondary and backscattered electron emission. In general as kv increases resolution increases, but the image becomes "harder". 2. Condenser Lens Setting Turning the COND. LENS dial clockwise increases the current and vica versa. A high setting indicates a stronger lens current, smaller spot size, lower intensity, better resolution and a grainier image. Usually for low magnification and easier viewing a setting of around 300 is used, while for higher magnification photography a setting of close to 500 (and possibly higher) is more appropriate. 3. Objective Aperture The objective aperture is located at the base of the column to the right of the specimen chamber. Each aperture position is indicated by a line on the side of the aperture assembly and each aperture (including no aperture) can be selected by rotating the knurled knob at the end of the assembly. There are four aperture sizes: 100 um, 200 um, 300 um, & 400 um, corresponding to the lines numbered 4, 3, 2, & 1, respectively. The smallest size provides the best resolution and depth of field but also the lowest intensity image. The largest size provides a bright image but at the expense of resolving power and depth of field. 4. Astigmatism Adjustment Astigmatism is a very important parameter with respect to image quality and the image needs to be adjusted to remove it on an ongoing basis, particularly at high magnification. There are a few tips that Page5
will help in the process, but there is no substitute for perseverance here. To check whether astigmatism is present select an area of the specimen that has fine detail with high contrast at high magnification (around X 10,000 to X 20,000). Vary the fine focus setting about true focus and observe whether or not the fine detail shifts laterally in opposite directions as you go through focus. If this occurs, adjust the X and Y STIGMATORS independently until the best image results. G. Photography With the Quartz PCI Follow the Quartz PCI instructions on the separate sheet for capturing and obtaining an image after consideration of the following points. Use the full image and reduced area features to optimize image quality. You may wish to monitor contrast and brightness on the computer screen using the slow scan (one arrow) button on the VIEW panel before actually capturing the image (In this mode, only about 3/4 of the image height is used when using 1024 X 768 resolution). When capturing, use the PHOTO switch to record the finished image and make sure to click the STOP button on the PCI side panel to avoid overwriting the image. G. Accessory Features Gamma Control Excessive contrast of the image can be reduced by an accessory to the contrast control called the gamma circuit. This allows the low-level signals (dark area of the image) to be increased proportionately more than the high-level signals (bright areas of the image). Electronic Image Rotation This feature provides a method to compose the image within the frame guidelines delineated by the taped area on the left viewing CRT. It is also necessary when producing 3-D images. To rotate the image, flip the ROTATION switch on and rotate the dial as necessary. Polarity Reverse This feature allows you to produce projection transparencies directly on the computer screen, or onto 35 mm film for stereo slides. In general the image contrast setting for transparencies should be slightly greater than that used for negatives. To reverse polarity simply press the POLARITY button. Dynamic Focus Tilting a relatively flat specimen at low magnification (less than approximately X 2,000) often leads to depth of field problems because of the height difference between the top and bottom of the image. The dynamic Page6
focus can correct this by varying the focal plane as the raster scans the image. This control has not proven too useful for biological specimens. Electronic Image Shift At higher magnifications it is sometimes desirable to translate the image by small increments. The IMAGE SHIFT dials located beside the crt screen can be used to electronically move the image a few microns in both X and Y directions. Photographic Scan Speed Exposures are taken line by line as the raster moves across the screen. The slower the raster moves, the sharper the image on the negative. For normal work we use the #2 setting. Split Screen/Dual Magnification Display Unit To obtain dual magnification of the image, push the SPLIT/FULL switch in to divide the crt screen into top and bottom halves. Choose the degree of magnification you want for the high mag image (X2, X5, or X10). To select the most appropriate high mag. region, rotate the X and Y controls. This feature can also be used too obtain both secondary electron and backscattered electron images on a split screen, with the top half showing a secondary electron image and the bottom half showing a backscattered electron image. Before you do this, follow the instructions given below for using the backscattered electron detector. H. Other Operating Modes Backscattered Electron Microscopy 1. Bring the specimen chamber up to atmospheric pressure, place your specimen on the stage, insert the backscattered detector and align it to the specimen position (usually no higher than 5 mm from stub surface. 2. Pump out the chamber, open the gun valve, and obtain a secondary electron image as usual (use # 1 aperture). 3. Once a focused image is obtained, turn on the PM.HV toggle switch on the backscattered image control panel, and make sure the RE button is in and the polarity button is out. 4. Push the SE/EXT button in and leave the SPLIT/FULL button out. 5. Using the BRIGHTNESS and CONTRAST controls on the backscattered image control panel, optimize the image for photographic quality. 6. If you wish to obtain a split screen image of both types of images, push the SPLIT/FULL button in and further adjust parameters for both types of images by switching between SE and EXT settings. Page7
I. Shut Down 1. Standby If you need to leave the instrument for a few minutes make sure that you leave it in slow scan (three arrows) mode. If you leave for longer, desaturate the filament, turn the high voltage off, and close the gun airlock valve. For long periods of absence the DISPLAY should also be flipped off. If you expect the next user to arrive shortly, you can leave the instrument on STANDBY condition as set out in (A) above. 2. Turning Off J. Problems To shut the instrument down completely, you should remove your specimen and repump the column to the point when the green airlock valve lights up. Then: 1. Turn the MAINS switch off. 2. Wait at least 15 min. and then turn off the water supply. If you experience a problem while operating the SEM, you should contact me. From time to time the instrument malfunctions and damage may result if you continue to operate it under these conditions. Alternatively, you may be doing something wrong that can be quickly corrected with a little informed advice. Whatever the problem is, do not attempt to go beyond your level of expertise in trying to correct it. Remember the entire class as well as several research projects depend on a properly functioning SEM. Page8