User Guide – Infrared Microscopic Imaging
This guide outlines the steps required to collect infrared (IR) spectromicroscopic images using the Bruker Vertex 70v Interferometer, the Hyperion 3000 IR Microscope and the Focal Plane Array (FPA) detector.
Bruker Vertex 70v Interferometer / Hyperion 3000 IR Microscope
Beamline staff will introduce you to the instrument and controls during your initial Beamline Specific Orientation. A Visual Glossary of components and controls is also provided for quick reference.
|Hyperion 3000 IR Microscope||Optical Beampath of Microscope|
|For more details, see the Visual Glossary|
The steps in this section will have been done for you already by the beamline staff. However, you may find it necessary to restart OPUS at some point.
- Start OPUS 7.2 using the icon on the desktop.
- Open the Detector selection window and press `FPA`.
- Open the Advanced Measurement window, press the `Load` button and select the `HYPERION-3000-FPA-TRANS.XPM` file (or `_REFL.XPM for reflectance mode).
- Switch to the `Check signal` tab and wait for an signal value to appear.
- Close the Advanced Measurement window.
Liquid nitrogen (LN2) Detector Cooling
The detector will be initially filled with liquid nitrogen for you by beamline staff, however you will need to plan your experiments to accommodate refilling the detector. If you are unsure how to fill the detector or have never done it, ask a member of the beamline staff to demonstrate. You should also have completed the cryogenics safety training module.
The initial fill will last for approximately 6 hours. Subsequent fills last around 8 hours. It is important not to allow the detector to warm up while it is powered on, and especially during data collection. The detector cannot be refilled during data collection, so you will need to plan your experiments accordingly.
The purge system on the microscope stage reduces spectral interference from infrared-absorbing atmospheric gases such as water and CO2. Sample loading procedure:
- Mount your sample onto a sample plate. Use tape to secure sample disc to the plate.
- Remove the plexiglas purge components and rotate the top objective out of the way.
- Place your sample onto the microscope stage and return the objective to the working position.
- Reassemble the purge system around your sample.
|Purge System: Open||Purge System: Assembled|
In OPUS, open the FPA wizard using the
Start video wizard button:
This will launch the microscope wizard and bring you to the
Select Device window. Choose
Hyperion 3000-FPA to select the FPA imaging detector and press
Next to continue:
Next again at the
Reset xyz stage window. Do not press the
Reset xyz stage button.
Using either the video display or the ocular viewer, bring your sample into focus and align the bottom (condenser) objective for optimal IR illumination using the following directions:
Note: To select between the video display and the ocular viewer, use the Ocular / Video Selector.
Set both the bottom and top apertures to the
OPEN position. Focus your sample using the microscope stage Z control in visible light mode. Illumination of the sample can be adjusted using the bottom illumination control.
Select a smaller bottom aperture (
0.9 mm in this example) and adjust the condenser stage Z until the edge of the aperture is in focus:
|Bottom Aperture: Out of focus||Bottom Aperture: In focus|
Return the bottom aperture to the
OPEN position. Using the ocular viewer, perform a final adjustment of the condenser stage Z to bring the edge of the aperture (at the very limit of the field of view) into focus. Ensure the aperture is centered in the field of view using the condenser stage XY controls.
You can now collect a visual image of the sample in order to select the regions to be measured with the IR microscope and to orient yourself on your sample.
objective settings are correct (Transmittance, 15x Objective in this case):
If you are only interested in the part of the sample already in the field of view, press
Single image. However if (more likely) you wish to survey a larger area of your sample, choose
Image of larger area....
Define Larger Area
Translate the microscope stage to a point at the extreme corner of the rectangular area you would like to survey. Note that the larger area you define, the longer this image will take to collect.
Once you have reached the first corner, press the
Add new border point button and the stage position will be saved.
Repeat with the opposite corner, thereby defining a rectangular area of your sample.
The software now shows the number of tiles that will be collected and the stage positions defined by your selection. If you are satisfied with this sample area, press
Collect defined image. Otherwise, remove the points and choose new ones.
When collection is finished, you are returned to the Collect visual images view. The visual image will be saved with your OPUS data file, but you can also now right-click and
Export currrent image... to save it separately. If you are happy with the visual survey, click
At this point you should collect an appropriate infrared background. You can choose to either collect this background once or every time after a set number of sample measurements has been completed.
User defined background position and press
The choice of background position is important. It should include the sample mount/window and any other material common to your sample mount which is not part of the sample itself.
If there is an appropriate background position within the visual survey image, you can navigate to that area now by selecting
Move xy stage as the Mouse mode and simply clicking on the desired location of the Overview image.
If, as in this example, your survey image does not contain an appropriate background, pan the sample stage using the Microscope Stage XY control joystick.
Once you are satisfied with the selected background location, press
Set background position. Note that the software will remember this position as well as your defined survey area for the duration of the measurement.
The collection of the background will define some spectroscopic parameters that must be consistent between the background and the measurement:
|Resolution||2? – 16 cm-1|
|Binning||None – 16x16|
The number of scans (Scan Time) is also selected here but this parameter is not required to match between the background and sample measurements. Further discussion of the consequences of binning can be found in Appendix A: Binning.
Ensure that measurement mode is set to
Transmission, Objective is set to
15 x IR and Microscope channel is set to
IR. You should now see something like this (4x4 binning):
The bottom-left image is the live FPA image, and the bottom-right is a plot of every FPA pixel (after binning). Note that the FPA image is evenly filled and centered, and that the FPA signal level (small white circles) is at approximately 4/5ths of the detector dynamic range (lighter blue rectangle). If necessary, you can rescale the FPA image to the max/min of the measured signal by double-clicking on the image.
When ready, press
Measure background. A prompt to check polarizers and condenser alignment will appear, giving you one last chance to center the signal on the FPA. Press OK and the background will be collected.
You can now select the sample positions to image with the infrared detector. In this view, you can select between IR and VIS modes. Navigation around the sample is again possible using either the Microscope Stage XY joystick or by clicking on the Overview image while in
Move xy stage Mouse mode.
Once you have located the desired imaging region, press the 2x2 blue grid button under Measurement positions. It will turn green and your cursor will change to FPA GRID when over the survey image. Click on the desired area and the Set number of measurement positions window will appear:
Here you can set the exact position and number of FPA infrared image tiles to collect. Press
OK and the selected grid of tiles will be shown on the Overview image. At this point you can drag the grid to reposition or right-click and edit the exact position and number of tiles. Press
Next when you are satisfied with the selected area.
Enter your Sample Name and choose a File Path in the
D:\Data\yourname directory where your data will be stored. The File Name variable can be edited to include only the Sample Name (
<snm>) or to include the date (
<Y><m><d>) or time (
<H><M>) as desired. You can also choose the sample Scan Time and the calculated total measurement time is shown.
You are now ready to begin collection. Press
Measure Sample and the defined measurement area will be collected.
Once data collection is complete, you will be returned to the Collect visual images window. You can now select
Repeat to open a new Chemical Imaging tab to allow preliminary analysis of the collected data. To continue imaging, select the Videobased FPA measurement tab and repeat the above procedure.
Once you are finished analyzing a dataset, right-click on it and select
Unload File. Too many open maps may cause OPUS to become unstable or crash.
Further discussion of the data analysis tools is beyond the scope of this guide.
When you are finished collecting data for your shift, you will need to:
- Close OPUS, ensuring any edits you have made to your data files have been saved
- Turn off the FPA detector using the toggle swtich on the detector itself
- Transfer your data files onto a portable storage device
- Tidy up any materials related to your sample / analysis
Microscope XY Stage control not responding
Open the XY Stage control and enable the
Enable Joystick checkbox setting.
No visible image in either ocular or video viewer
Press the visible light mode button, even if it is already selected. This problem is particularly common in reflectance mode.
No IR signal
Check that the detector is cold (recent LN2 fill) and that nothing is obstructing the IR beam.
Unable to collect Visual image using Define Larger Area
The XY stage may have become uncalibrated. Contact beamline staff to assist you in calibrating the stage.