eTraining Introduction

Specimen Preparation

Leica Ultracut UCT



Hitachi S-4700 FE-SEM

Hitachi FB-2000A FIB


Veeco Dim 3000 AFM

Fluorescence Microscopes



Focus Surface Icon1. Select the Focus Surface icon. NOTE: To move the stage, the Focus Surface menu must be open! VIEW
2. Move the stage using the trackball. To move large distances, hold the lock button and move the trackball. To make finer movements, use only the trackball. To adjust the vertical distance between the sample surface and the cantilever, depress the Focus button while using the trackball. NOTE: Make sure there is enough vertical clearance between the surface of the sample and the cantilever before moving the sample underneath the cantilever.
3. Position the stage beneath the cantilever and focus.
 3.1. A sample with surface topography clearly distinguishable under an optical microscope can be positioned directly beneath the cantilever. Adjust the cantilever so that it is positioned ~1 mm above the surface, and then focus.
 3.2. If the sample is transparent or smooth, position the sample so that the laser beam is near the edge of the sample. Adjust the stage so that the cantilever is ~1 mm above the surface of the sample and use the edge of the sample to focus.
 3.3. Focus can be achieved by depressing the Focus button on the trackball and moving the trackball either up (away from you) or down (towards you).
4. Move to a clean area of the sample where you would like to image and make sure the surface is still in focus.
5. Once the image is in focus, select OK. NOTE: An alternative sample loading procedure is to select Stage > Load New Sample > OK. This will move the stage in and out under the AFM. Always check to make sure the sample will not hit the cantilever before moving the stage.
6. Set up the scan parameters using the menu boxes. VIEW
 6.1. Scan Controls
  6.1.1. Scan Size: start with 1-5 Ám
  6.1.2. Scan Rate: start with 1-2 Hz
  6.1.3. Samples/ Line (resolution): start with 256
  6.1.4. Slow Scan Axis: enabled
 6.2. Feedback Controls
  6.2.1. Tapping Mode: determined by software/ previous user
  6.2.2. Contact Mode—Integral Gain: 2.0, Proportional Gain: 4.0 (twice the Integral Gain)
 6.3. Channels 1-3: An image will be produced for each channel that is turned on. To turn off a channel, simply select OFF. VIEW
  6.3.1. Data Type: at least Height (in Channel 1)
  6.3.2. Data Scale (Z-range): select for good contrast. If this value is too large, your image may not show any topographical features.
Engage Tip Icon7. Bring the tip in contact with the surface by selecting the Engage Tip icon.
Scope Trace Icon8. To optimize scan parameters, select the Scope Trace icon.
 8.1. Amplitude Setpoint: determines the force used to tap the surface in tapping mode.
  8.1.1. In the menu box, make sure that Amplitude Setpoint is highlighted. VIEW
  8.1.2. Looking at the right monitor, increase the setpoint by pressing the right arrow key on the keyboard until the scope trace indicates that the surface is not being touched (the status bar turns red). The two graphed lines should be horizontal on the X-axis. VIEW
  8.1.3. Press the left arrow key until contact is initiated (the status bar becomes white). The corresponding amplitude setpoint is referred to as A0.
  8.1.4. Multiply the obtained value for A0 by 0.8 (80%) and type the new value into the menu. Then hit ENTER on the keyboard.
 8.2. Adjust Scan Size, Scan Rate, and Data Scale until the two graphed lines are symmetrical.
 8.3. Adjust the Gains: Select Microscope > Auto Gain Adjust. Click OK once the software has suggested gains.
Return to Image Icon9. Once the parameters have been chosen, return to the image by selecting the picture icon.
10. Adjust the image to get the proper brightness and contrast by changing the Data Scale entry in Channel 1.
11. To capture an image, select Capture > Capture Filename, and give the file a name. NOTE: The extension .000 is added automatically and successive images will be given extensions .001, .002, and so on. VIEW
12. If the scan begins in the top half of the image, select the down arrow to begin scanning from the top of the image. Image From Top Icon If the scan begins in the bottom half of the image, select the up arrow to begin scanning up from the bottom of the image. Image From Bottom Icon Doing this makes the image capture faster and puts less strain on the instrument.
13. Select the Capture icon to begin image capture. Image Capture Icon To cancel a capture, select the Cancel Capture icon. Capture Cancel Icon The capture status appears at the bottom of the screen. When an image has been captured and saved, the status should read Capture: Done .
Withdraw Tip Icon14. When done scanning, select the Withdraw Tip icon.
15. Reposition the surface for a new scan by selecting the Focus Surface icon and use the trackball to move to a new position, or load a new sample by selecting Stage > Load New Sample. VIEW

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