Figure 1: Photograph of the 1.4.3 FTIR spectromicroscopy endstation, which consists of a Nicolet 760 FTIR bench (to the left), a Spectra-Tech Nic-Plan IR microscope (in the center), and video display and stage controller for the microscope (on the right).

Operation Checklist

• Cooling the detector
• Locating your sample
  
• Transmission Mode Setup
• Checking Signal Level (OMNIC software)
• Background Acquisition
• Sample Acquisition (point, multiple points, line, area)
  

Cooling the Detector

Please wear safety glasses and cryogenic gloves for this operation!

1. Fill green thermos labeled 'Nicolet' with liquid nitrogen from 4L dewar.  If there is no liquid nitrogen in the 4 L dewar, you may fill it at the user liquid nitrogen station.
2. Open funnel lid (gray flip top on blue plastic mount) on top right of microscope.
3. SLOWLY pour in liquid nitrogen.   When first beginning to fill, pour in only enough liquid nitrogen to fill the funnel (you will see it start to bubble against the plastic screen), and then stop pouring for at least 60 seconds and let that amount drain down the funnel. Repeat this two or three more times to fully cool down the funnel and tubing system. Then slowly pour the rest of the liquid nitrogen into the funnel making sure to never overflow the funnel.
   
4. The dewar will typically remain cold for > 10 hours, but you should keep track of when you filled it so that the detector does not get warm over a long measurement!

Locating your sample

1. Place your sample on the microscope stage.
2. Move the x-y stage using the joystick on its controller to place this sample directly below one of the microscope objectives. It is often easier to use the 10x glass objective to locate the region of interest before selecting the higher magnification IR objectives. 
   
3. Press the ‘view’ button on the front of the microscope to allow visual inspection of the sample. This button is a little flaky, but should work after repeated attempts (the green light will be lit).
   
4. Rotate the upper illumination adjuster (right knob at bottom of microscope) to turn on the upper light source.
   
5. Look either into the eyepieces or at the TV monitor and adjust the focus (using the coarse and then fine focus knobs looking for the brightest image. If your sample is on a gold substrate or another good reflector, you may need to lower the upper light intensity when you are getting close to the correct focus because the image will be too bright with the light at its maximum level. You can verify that you are focused on your sample by slightly moving the sample using the joystick on the x-y stage controller and you will see the image move. If it does not move, you have not yet found the sample focus.
6. If you are doing reflection mode, you are now ready to check the signal level. If you are doing transmission mode, continue on to Transmission mode setup.
   

Transmission Mode Setup

1. Insert the circular aperture with a label matching the objective you are using (15x or 32x) into the upper location on the microscope (see Figure 2). Lower the black plastic flap in front of the aperture location and slide the appropriate aperture into place taking care to have the tape label on the aperture facing out. This will define a 100-micron area on your sample that we will use to align the lower objective. You should now see the 100-micron area with the rest of your sample masked away on the television monitor or in the microscope eyepieces. If you do not, check that the upper light source is on and the pellicle beamsplitter (silver knob located just below aperture on right side of microscope) is rotated toward the back.  If your sample is not visibly transparent, then move the sample stage to the open hole using the joystick on the sample stage controller, otherwise just leave the sample where it is and turn down the upper light source intensity.
2. Insert the lower (1.0 mm) aperture into its position (see Figure 3 ). This time make sure the tape label is facing the rear of the microscope. This will again make a 100-micron area illuminated at the sample stage.
   
3. Increase the lower light source intensity (left knob at bottom of microscope), and focus the lower objective by using the vertical (z) knob of the x-y-z translation stage located on the left side of the microscope (see Figure 4). Adjust this knob until you see on the television monitor or in the eyepieces a clear circle of light.
4. Adjust the upper light source and lower light source so that you can see both clearly.
5. Align the lower objective with the upper objective by using the x and y knobs of the x,y,z translation stage.
6. Remove the alignment apertures and check the signal level.

Figure 2. Photos showing where the upper aperture goes, and on the right with the aperture inserted. Make sure the label tape is facing out as the thickness of the tape would misalign the aperture if it is facing the alignment bars on the inside.

Figure 3 . Close-up photograph of the lower objective showing where the lower aperture is inserted. The lower focus know is also seen and labeled (it has a piece of light green tape on it), and the lower objective position adjusters are barely visible at the top of the images.

Figure 4 . Lower objective translation stage.

Checking Signal Level

The spectrometer and microscope are controlled through OMNIC.  For more detailed information, please see the OMNIC manual.

1. Open OMNIC and logon with your user ID.
2. After selecting Experiment Setup from the Collect menu, choose the Bench tab to display the live interferrogram.
   
3. Check that the sample compartment is set to either 'Right μscope '%T' or 'Right μscope %R' depending on if you intend to do transmission or reflection.
   
4. Check that the source is set to 'External' if you plan to use the synchrotron.
5. Click Peak to Peak and look at the value of the interferrogram.  For the 32x objective, reflection on gold typically gives a peak to peak value of ~6.5 (500 mA ring current, Gain 1), whereas transmission through air gives a peak to peak value of ~12. If you do not see an interferogram, please look at the troubleshooting page.  If the transmission signal is low, you can fine tune the position of the xyz translation stage to maximize the signal.

Figure 5. Experiment setup window with the 'Bench' tab selected. Typical values and selections are shown.

Background Acquisition

1. After selecting Experiment Setup from the Collect menu, choose the Collect tab to display the collection parameters. For a detailed explanation, please go here.
2. Change the resolution and number of scans to the desired quantity.  Typically, you will take the same or greater number of scans for your background as for your sample.
3. Select 'Collect background after ____ minutes' and press OK.
4. Position your reference material (air, gold, substrate, etc.) under the IR objective.
5. Select Collect >> Collect Background...
6. Select File >> Save as to save the background in your directory for future use.

Sample Acquisition

Single Point Spectrum

1. After selecting Experiment Setup from the Collect menu, choose the Collect tab to display the collection parameters.
2. Change the resolution and number of scans to the desired quantity. Note: the resolution must be the same as the background file you plan to use.
3. Select the background handling method. If you choose 'Collect background after ___ minutues', then the background will be the most recent one taken.  If you prefer to use a saved background, you can select 'Use specified background file:' and click 'Browse' to locate your background file.
4. Select Save and then press OK.
5. Press View on the microscope to enter view mode.
   
6. Select 'Show Atlus window...'  from the Atlus window to see your sample on the computer
7. Posiition your sample under the red crosshairs using either the joystick or the Stage movement tool.
8. Select Collect >> Collect Sample... This will collect a spectrum at the current location of the red crosshairs.
9. Select File >> Save as to save the spectrum in your directory.

Collecting Maps (multiple points, line, area)

1. Select 'Show Atlus window...'  from the Atlus window to see your sample on the computer. You may need to press View on the microscope to enter view mode.
2. Make sure that the stage calibration file is correct for the objective you are using. The Atlus window title will say the name of the objective.  If you need to change it, then select the calibration bar icon (located in the middle of the lower Atlμs toolbar) and change the calibration file to the appropriate objective.
3. Select the points, line, or area of interest by using the mapping tools in the lower left hand corner of the Atlus window. You can draw your map on the video screen.
4. Change the step size by selecting the 'Mapping Tab' after selecting Collect >> Experiment Setup. The estimated time will be updated after you press 'Apply'.  WARNING: a scan often takes ~1.5 times the estimated time!  To adjust the time, you can change the step size, the number of scans, or the size of the map.  This may take several iterations of looking at your sample and changing the parameters before getting a reasonable map time (recommended < 8 hours).
   
5. Under the Collect tab after selecting Collect >> Experiment Setup, choose the background handling method to be 'Use specified background file:''  and choose your file.
   
6. Press Save and OK.
7. Press View on the microscope and adjust the microscope illumination to optimize the image on the computer.
8. Select Collect >> Collect Map. You will be prompted for a filename and then the software will take a picture and begin collecting your map.