Standard Operating Procedure

- Renishaw inVia Micro-Raman Microscope

Susheng Tan, Ph.D.
NanoScale Fabrication and Characterization Facility, University of Pittsburgh
M104 Benedum Hall, 3700 O’Hara St., Pittsburgh, PA 15261
Phone: (412) 383-5978, Email: [email protected]

Log on the Raman Microscope via FOM in any networked computer.

I. System Start-up
1. Check and make sure the main system unit (orange spectrometer) is ON.
2. Power on the PRIOR stage controller for the motorized xyz stage.
3. Power on the desired laser(s):
a. 229 nm and 488 nm lasers: only started by NFCF staff, indicate your need when you reserve
the instrument time.
b. 633 nm: switch on the key on the laser unit
c. 785 nm laser: switch on the key on the laser unit
4. Start the Renishaw WiRE 3.0 software.
a. The software will prompt for a position check of the relevant motors.
b. Choose the ‘Reference un-referenced motors only’ option, and click on ‘OK’.

II. Quick Calibration on Si (100) Reference Sample

1. Open Measurement>New Measurement.
2. Select “Internal Si Reference Measurement” (with the same laser you use) and then click OK.
3. Use Measurement>Run to collect static Raman spectra of Si(100) at 520 cm-1.
4. Zoom into the peak at 520 cm-1. Right click to bring up options to check peak position. If it is not in the
520-521 cm-1 range, calibrate the system by clicking Tools>Calibration>Quick Calibration.
Alternatively you can calibrate the Raman shift by clicking Tools>Calibration>Offset and input the
offset value (positive if the Si peak is greater than 521 cm-1).
5. Repeat steps 3-4, if necessary.

III. Collecting Spectra on Samples

1. Click “View the Sample under Video and Laser” icon on the bottom menu bar.

2. Load your sample (on microscope slide) to the stage and focus on the surface.
3. Focus your specimen using the microscope focus knobs. If your specimen is featureless, you may
reduce the field stop and focus with the focus stop as shown below.

4. Move the Region of Interest (ROI) to the center of cross hair to which laser is positioned.

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 5. Use Measurement>New>Spectral acquisition or to start a new spectral measurement. Modify the
“Spectral acquisition Setup” for your experiment following the setup guidelines:
5.1. Range tab:
Grating Scan Type: Extended.
Spectrum Range: Low 100; High 3200 and
Raman shift should appear in the white box to
the right.
Configuration: (no lenses change is needed for
visible wavelength).
Laser Name Grating Name
785 nm (300 mW) 1200 l/mm
633 nm (17 mW) 1800 l/mm
488 nm 1800 l/mm
229 nm 3600 l/mm, UV optics

5.2. Acquisition tab:

Exposure time: 10s
Accumulations: 3
Laser Power: 10% (sample dependent)
5.3. File tab:
File name: -Browse >Desktop >User Data > (Your folder) > Enter a file name to which the Raman
data will automatically be saved.
Auto Increment: Selected (This option will automatically save each subsequent run to the entered
file name incrementally. (i.e. If your file name you entered is called “Raman” then each subsequent
run will be saved as “Raman1”, “Raman2”, “Raman3” etc.)
5.4. Unless the sample has some pre-determined needs, no adjustments need to be made under any of
the other headings. To apply your settings, click either “Apply” then “OK” or simply click “OK”
6. Use Measurement>Run or the Run button to collect spectra.
6. 1. Change the “Grating scan type” to static with the spectrum centered at a strong peak.
6.2. Go to Measurement>Cycle to collect static LIVE Raman spectra of your sample and use Leica
focus knob to optimize signal to maximum counts.
7. Use Measurement>Run or the Run button to collect spectra.
8. Save the spectrum if you haven’t set the automatic save.

IV. Mapping on Samples

1. Follow the same as in Section III.
2. Go to Measurement>New>Map acquisition to start a new mapping measurement and to setup acquisition
parameters for your experiment.
a. Under the Image Source section, ensure that "Video Viewer" is selected.
b. In the top tool bar, left click once on the leftmost icon to display a drop-down menu. Using this
menu, you may select the shape of the area you wish to map.
Note: The term "filled" in the menu indicates that the instrument will scan points both on the
border of the shape and inside the shape. For a shape that does not specify "filled", only
the outline of the shape will be scanned.
c. When you have selected your shape, then move the mouse to the video viewing section of the
screen and draw the shape on the image by left clicking on the mouse, holding, and dragging to
make the shape the direction and dimensions that you want.
Note: If you are unsatisfied with the placement or size of the shape, you may remove it and try
again by clicking the "X" icon in the top toolbar of the "Map Points" dialog box.
Note: The units for the step size are not specified in the dialog box, but they are microns.

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 Note: Between the "Area Properties" section and the "Image Source" section, there is an
information section that will indicate the number of points in the scan. The information in
Table 1 may be used as a guide for setting up mapping runs.

Table 1. Run time information based on Area and Step Size.

Time (hours) Area (microns) Step Size (microns)
2 50x50 5
7 50x50 2.5
11 50x50 2
28 100x100 2.5
7 100x100 5

3. When you are satisfied with the parameters, click "OK" and the "Map Measurement Setup" dialog box
will automatically appear. You may now set up the parameters for the spectral acquisition as described
in the Spectral Acquisition Setup.
4. Use Measurement>Run or the Run button to collect mapping spectra.

V. Mapping Analysis
1. After you have finished the map acquisition, open the map data file by going to File > Open or File >
Open in a New Window, or by clicking on the Open icon in the toolbar.
2. In the Navigator window click on the Data tab and make sure that the data file you wish to analyze is
highlighted.

Figure. Location of “Data” tab.

3. Go to Analysis > Mapping Review and the "Map Selection" dialog box will appear. Select the type of
map you wish to create. Then click on the "Create..." button.

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 Note: The most common type of map is the "Intensity at a Point". This map allows you to select a
given wavenumber and generate a map that displays the relative intensity of that peak at each
point. This is the kind of map that will be used as an example for this guide, however, the setup
for the other types of maps is very similar.
4. After hitting "Create", a dialog box will appear. You may use the mouse to left click (once) on the
vertical black line and drag it to any point in the spectrum. If necessary, you can zoom in on an area as
you would normally. When you have selected the desired point, click on the "Create New Map" icon
(second from the bottom on the right-hand side).
5. You will return to the "Map Selection" dialog box automatically. To view the map you have created,
click "View"
a. While viewing the map, you can click on any point to display the complete spectrum at that point
(Bottom right), the Intensity at selected wavenumber compared to other points in the same Y-line
(Upper right) and the Intensity at selected wavenumber compared to other points in the same X-line
(Bottom left).
b. You may save this data by going to File > Save As
c. You may save the view on the screen by going to File > Save View As

VI. Shutting down

1. Close the WiRE 3.0 Software.
2. Shut down the PRIOR xyz stage controller.
3. Turn off the lasers if nobody else reserved the Raman immediately after you.
5. Turn off power of the spectrometer
6. Leave computer ON and turn off the monitor.
7. Sign off the instrument via FOM.

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