FT-IR Microscopes

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Introduction

The Thermo Scientific Imaging ATR (Attenuated Total Reflection) accessory for Nicolet™ iN10, Nicolet iN10MX and Nicolet Continuum™ FT-IR microscopes supports chemical imaging studies with enhanced spatial resolution from a single, large area contact point. The large contact area allows for quick collection of area maps and images. It is ideal for mapping samples that may be disturbed or deformed by using multiple contact points, such as paints, cultural artifacts, biological tissues, and sticky or oily materials. The accessory is generally not applicable for liquids and loose powders.

This document covers:

• Operating Precautions
• Features and Operation
• Installation
• Operation
• Step-By-Step Example Using OMNIC Picta Software
• Maintenance
• Appendix A - Total Force Applied by the Pressure Column

Conventions Used in This Manual

This manual uses the following conventions for providing safety and other special information:

• NOTICE: Follow instructions with this label to avoid damaging the system hardware or losing data.
• Note: Contains helpful supplementary information.
• Tip: Provides helpful information that can make a task easier.

Operating Precautions

NOTICE: The germanium crystal can be broken if small, hard materials are compressed against it at maximum pressure.

To maximize the life of your Imaging ATR accessory, heed these precautions:

• Do not apply pressure to the crystal without a sample in place.
• Do not use the pressure device to crush a sample.
• Make sure your sample will not react with the germanium crystal.
• Do not rub the crystal against the sample.
• Do not scrape the crystal with any type of metal tool.
• Do not use abrasive cleaning agents, pads or strongly acidic or basic solutions to clean any surface of the accessory.

Features and Operation

The Imaging ATR accessory is designed to support chemical imaging studies with enhanced spatial resolution. Micro-ATR techniques place the sample in direct contact with a germanium ATR crystal, resulting in improved spatial resolution compared to open beam transmission or reflection measurements in air. Single point ATR mapping with a slide-on micro-ATR accessory offers similar benefits and greater flexibility in image size and dimensions. However, single point mapping requires many separate contact points to build an imaging dataset. The Imaging ATR accessory is a faster alternative as it requires only a single, large area contact point.

Components

The key components are:

1. Baseplate: Mates to the standard 3 3/8 in diameter sample holder recess found on all Thermo Scientific infrared microscope stages.
2. Stage Lock Lever: Activates a cam to secure the accessory, preventing rotation or shifting that may affect results.
3. Crystal Arm: Holds the anti-reflection coated germanium ATR crystal. If the crystal is damaged or broken, a new crystal arm can be ordered and replaced by the user. Refer to Maintenance for details.
4. Crystal Pivot Screw: A hex head machine screw, covered by a cap, that is removed to change the crystal arm.
5. Crystal Arm Lock: A detent indicates when the crystal arm is in the proper position for data collection. It ensures the crystal arm does not move under pressure. Tighten the knob finger tight to lock the arm during operation.
6. Stainless Steel Pressure Column: Holds the sample and presses it upward against the ATR crystal to achieve optical contact.
7. Pressure Column Centering Screws (2): Used to bring the sample directly onto the optical axis. The sample should be placed as close to the center of the pressure column as practical.
8. Storage Disk: Must be installed in the sampling area under positive pressure when the Imaging ATR accessory is not in use. It maintains proper operation of the hydraulic assembly and protects the germanium crystal from being scratched by metal parts. Refer to Installing the Storage Disk for details.
9. Pressure Column Cap: Can be placed on top of the pressure column to provide a larger, more stable sampling surface. Remove the cap to accommodate thick samples.
10. Sample Holders (3): Hold the sample in place during analysis. The recess in the top of the pressure column accepts 13 mm diameter disks. Custom sample holders may also be used. Mount your sample as close to the center of the sample holder as practical.
11. Pressure Column Control Knob: Rotate clockwise to raise the pressure column and counterclockwise to lower it. This control manages the fluid-driven assembly to expand or compress the column spring. An internal clutch protects the hydraulic assembly from over-pressurization. Refer to Using the Pressure Column Controls for details.
12. Pressure Indicator: Monitors the internal pressure of the drive assembly. A negative reading indicates the pressure column is compressed; a positive value indicates it is expanded.
13. Pressure Indicator Battery Access: Provides access to the 3-volt lithium coin size battery for the pressure indicator. Battery life is estimated at 7 months of continuous use.
14. Pressure Indicator On/off Switch: Use this switch to turn off the display to extend battery life. Press momentarily to change between on and off states.

NOTICE: If you press and hold the on/off switch for 5 seconds, the pressure indicator records the current pressure as zero and reports changes relative to this new zero state. Avoid operating in this state as the absolute internal pressure is no longer known. To restore factory settings, remove and then reinstall the battery.

The kit also includes sample spacers in several thicknesses, tweezers for manipulating spacers or 13 mm disks, and self-adhesive polyimide masking discs for practice samples or performance checks.

Using the Pressure Column Controls

NOTICE: Handle the pressure column controls carefully and follow the instructions below or you may damage the crystal.

Before placing a sample on the crystal, turn the pressure column control knob counter clockwise to lower the pressure column to prevent scraping or rubbing the crystal against the sample when rotating the crystal arm.

When the column is retracted, the pressure reading is negative. The safe value depends on sample thickness. When fully retracted (for the thickest sample), the indicator typically reads between -10 and -12. Thin samples may not require full vacuum for clearance. After establishing a safe indicator reading for a sample type, you can use the indicator alone without visual checking for clearance.

After loading the sample (see Mounting and Loading a Sample), turn the pressure column control knob clockwise to raise the pressure column. The indicator becomes less negative and then swings to positive values as contact is made with the crystal. Once contact is made, the pressure column no longer expands, and internal pressure builds rapidly with small turns of the control knob.

Sample thickness influences the indicator reading for clearance and the absolute reading upon contact. Once this contact point is established for a particular sample type, you can use the indicator reading to quickly re-establish the same contact pressure for similar samples.

The maximum safe internal pressure is 40 psi. For most samples, good optical contact is achieved at low pressures (2 to 15 psi). The pressure control knob has an internal clutch to prevent gross over-pressure conditions, with a slip-point far above typical sampling pressure. Always use the digital indicator, not the clutch slip-point, to monitor sample pressure.

Tip: The pressure indicator reports the internal pressure of the hydraulic fluid, not the pressure applied to the sample. Its purpose is to ensure operation within safe limits and allow reproducible settings for retracted and contact positions. Consider the indicator to report arbitrary units.

Specifications

^a 400 x 400 micrometers on the Nicolet Continuum microscope

Installation

Contents

• Installing the Accessory
• Installing the Storage Disk

Installing the Accessory

Follow these steps to install the Imaging ATR accessory in your FT-IR microscope.

To install the Imaging ATR accessory

1. Lower the condenser on your microscope all the way, or demount it if applicable (see "Tips for specific Thermo Scientific microscopes" below).
2. Lower the microscope stage far enough to accommodate the accessory (typically near its lower limit). It may help to move the stage as far to the left and forward as possible to make more room. Refer to your microscope's documentation for help with these operations.
3. Place the accessory in the round recess of the stage top plate.
4. Rotate the accessory in the stage until it is square with the front top plate of the stage.

5. Activate the stage lock lever to keep the stage firmly in place when in use. Move the lever clockwise until it touches the post.

Tips for specific Thermo Scientific microscopes:

• Nicolet iN10 family microscopes: The Imaging ATR accessory always operates in reflection mode. Use the IR Energy tool to control the condenser and stage motors. Use the Park Condenser button to move the condenser to its lower limit. The Eject Stage button automatically lowers the stage and moves it to a convenient location for mounting. To control stage height, use the IR Energy tool's slider or the Focus control on the virtual joystick.
• Nicolet Continuum microscopes: The condenser is normally mounted or demounted for different magnifications. It must be demounted when using the Imaging ATR accessory (place the condenser in a clean, safe location). To control stage height, use the motorized Focus control knob on the right side of the microscope, or the optional focus control in the Atlus window if Atlus software is installed.

Installing the Storage Disk

The Imaging ATR accessory includes a red plastic disk that must be installed in the sampling area under positive pressure when the accessory is not in active use. The disk is required to maintain proper operation of the hydraulic assembly and protects the germanium crystal from being scratched by metal parts.

Install the disk when you are finished analyzing samples for the day, and leave it in place if you remove the accessory from the microscope.

NOTICE: Do not store the Imaging ATR accessory at vacuum, that is, with a negative reading on the pressure indicator.

To install the storage disk

1. Lower the pressure column by turning the control knob counter clockwise.
2. Release the crystal arm lock and swing the arm out of the way.
3. Remove any sample or spacer from the pressure column. The pressure column cap may remain in place if installed.
4. Place the red plastic storage disk on the pressure column.

5. Close and lock the crystal arm.
6. Rotate the pressure column control knob clockwise until the indicator reads about 20 psi. The exact value is not important, but do not exceed a reading of 40 psi.

Operation

Note: If the accessory has been stored under negative pressure, the pressure column may no longer retract. To restore normal operation, install the storage disk and leave the column under pressure overnight.

Contents

• Preparing the Accessory for Operation
• Mounting and Loading a Sample
• Achieving Optical Contact
• Collecting Spectra and Maps
• Using the Map Math Process Function
• Video Calibration with OMNIC Picta Software

Preparing the Accessory for Operation

Perform these operations before beginning to use the imaging ATR accessory:

• Center the accessory and the ATR crystal
• Center the pressure column.

Centering the Accessory and the ATR Crystal

The purpose of this step is to place the apex of the germanium hemisphere crystal on the optical axis of the microscope. One method is to lower the stage to its maximum extent and move it until the top of the crystal is in the middle of the field of view. Because the crystal surface is curved, this can be challenging. On some microscopes, the stage may not lower far enough to bring the top of the crystal into focus. The following is an alternative, preferred method.

To center the accessory and the ATR crystal

1. Press the Home button of your stage control software. Assuming your stage is correctly initialized, this places the center of the stage recess, and thus your ATR crystal, very close to the correct x-y location (directly below the center of the microscope objective).

In this document, x refers to the right-left axis, y refers to the front-back axis, and z refers to the up-down axis.

2. Position the stage near its lower limit and monitor the reflected IR energy as you raise the stage (energy will increase). Adjust the stage height so the reflected IR energy is at its maximum value. (Watch the interferogram intensity in a real-time display, e.g., on the OMNIC Experiment Setup > Bench tab, or use the IR Energy control in OMNIC Picta software.) This will be close to the correct focus (z) position, but the crystal is still not centered on the optical (x-y) axis.
3. With the reflected light illuminator set to maximum, move the stage up to 500 micrometers in the x and/or y direction until you see a brightly reflected circle of light on the video display or through the eyepieces.
4. Adjust the stage height to bring the edge of the circle in sharp focus. At this point, the image should look similar to Figure 5.

5. Move the center of the field of view to approximately the center of the bright circle. You can maximize the focus again by adjusting the stage height and observing the IR energy, but it should be close.

Tip: At this point, it is helpful to place a background point in the center to mark the optical axis (use the Map Background Point tool in OMNIC Picta or Atlus software). This allows you to use the Move To Background feature later to quickly return to this position.

Centering the Pressure Column

After centering the crystal on the optical axis, you must also center the pressure column.

To center the pressure column

1. Release the crystal arm lock and swing the arm out of the way so you can see the top of the column.

Note: Do not move the microscope stage x-y controls; the accessory must remain on the optical axis.

2. Remove any sample and/or spacer from the column.
3. Raise the column and/or focus so that the top of the column (or column cap) is in view.
4. Adjust the pressure column centering screws so that the obvious concentric machining rings lead you to the center.

The crystal and column are now aligned.

Mounting and Loading a Sample

To mount a sample

Samples can be mounted in many ways. A common method is to place the sample at the center of a sample spacer (included). Spacers are provided in several thicknesses to accommodate thin or thick samples. Use appropriate adhesive, double-sided tape, clay, or "blue-tack" to secure the sample if necessary, to keep it from moving during analysis.

The pressure column cap fits over the pressure column, providing a larger, more stable surface for holding most samples. However, the cap reduces the space between the pressure column and the germanium crystal. If more space is required, remove the column cap.

Tip: Place the region of interest as close to the center of the spacer as possible.

To load a sample

1. Use round-tipped tweezers to lift the spacer and place it into the 13 mm recess on the top of the pressure column or the pressure column cap.
2. If you moved the microscope stage for sample loading, return the stage to the optical axis (using the stored background point mentioned earlier, for example).
3. View the sample and use the centering screws to make fine adjustments to place the region of interest on axis.

Achieving Optical Contact

Optical contact is the most important consideration for achieving good results with the Imaging ATR accessory. For area maps, the entire region of interest must be in optical contact with the crystal surface. Because ATR is a surface-sensitive technique, any air gaps, even on the submicron scale, will lead to data loss. Therefore, the sample must be very flat (by polishing or cutting) or conform to the crystal surface under pressure. Particulate contamination on the sample or crystal surface can also create air gaps, so work in a clean area.

The Imaging ATR accessory can generate high pressures. However, higher pressure does not necessarily result in more complete contact; deformation of the sample by high pressure can result in poorer contact. Well-prepared samples of common materials like polymers, paper, and biological tissues should achieve good contact at pressure indicator readings below 15 psi. Harder samples may require more pressure. If good contact (indicated by a strong absorbance signal) is not achieved at a reading of 40 psi, there is likely an issue with the sample presentation. Inspect the sample and crystal surface for cleanliness or uneven surfaces preventing intimate optical contact.

Collecting Spectra and Maps

This section explains how to use the Imaging ATR accessory to collect a single point or map.

Note: To maximize the life of your Imaging ATR accessory, install the storage disk when finished using it for the day. Refer to Installing the Storage Disk for instructions.

Single Point Collection

The Imaging ATR accessory is primarily intended for collecting imaging data sets. It provides high-quality ATR spectra of single contact points and can complement slide-on micro-ATR tools.

To measure a single point

1. Collect the background before making contact with the sample.
2. Without moving the microscope x-y stage, raise the pressure column until contact is achieved, then collect the sample spectrum.

Collecting Maps

The Imaging ATR accessory is useful for collecting line and area maps using the mapping features of OMNIC Picta and Atlus software. When collecting imaging data sets, consider these concepts:

• The actual field of view on the sample is approximately 1/4 the size of what you see through your camera or eyepieces, meaning you are sampling a region that is 1/4 the size of what you see in air. (Refer to Video Calibration with OMNIC Picta Software for more information.)
• As the stage moves the crystal off the optical axis during mapping, the IR light travels at different angles through the crystal. This makes a single background spectrum inappropriate unless the mapped area is small. For large mapped areas, collecting a separate background for each stage position is ideal. Collecting maps in the Single Beam Spectra final format allows flexibility in applying appropriate background files and lets you collect background data before or after the sample data set.

Using the Map Math Process Function

The Map Math feature allows you to divide two maps with the same dimensions and coordinates. For example, you can use Map Math to divide two single beam maps to produce a map of ratioed spectra. Map Math is located in the Atlus menu in OMNIC software (if installed) and in the Analyze group in OMNIC Picta software. Refer to the Atlus or Picta Help for more information.

Step-By-Step Example Using OMNIC Picta Software

Video Calibration with OMNIC Picta Software

The video calibration in OMNIC Picta software is fixed and does not account for the effective magnification of the ATR crystal. When viewing the sample directly with the crystal arm open, software tools operate normally. However, when the crystal arm is closed, the effective magnification of 4x makes the indicated calibration on video panes 4 times larger at the focus. This affects software interaction:

• Aperture setting is magnified: A 100 micrometer aperture setting becomes a 25 micrometer image at the sample, allowing large apertures for good spatial resolution.
• Mapping step size is reduced at the sample: A 40 micrometer step size becomes a 10 micrometer step size at the sample, allowing larger step sizes for the same spatial resolution.
• Display area is magnified: Drawing a 1000 x 1000 micrometer area map results in a 250 x 250 micrometer area at the sample focus.
• Stage movement is reduced at the sample: When using the Move Stage tool, stage travel is reduced by 4x at the sample. Clicking on video or mosaic video panes moves the stage by the indicated distance, but it is 4 times less at the sample.

To prepare the accessory and measure a sample using OMNIC Picta software

This section guides you through preparing the Imaging ATR accessory and collecting background and sample area maps, then ratioing and saving them using OMNIC Picta software.

Note: Similar commands and procedures are also available in Atlus software.

1. In Experiment Setup, set the following:
   Mode: Reflection
   Final Format: Single Beam
   Collect Background after __ minutes: 0 (manual background control)
   Focus Handling: Do Not Focus During Map Collection
2. Click Home.
3. Activate the IR Energy control.
4. Click Park Condenser.
5. Manually lower the microscope stage until the indicator is near its lower limit.
6. Click Eject Stage. This saves the low position as the provisional sampling position.
7. Install the Imaging ATR accessory. Refer to Installation for instructions.
8. Follow the steps in Preparing the Accessory for Operation to center the accessory, crystal, and pressure column.
9. Open the crystal arm and place the sample spacer on the pressure column or pressure column cap.
10. Lower the pressure column so there is clearance between the sample and crystal when you swing the crystal arm.
11. Close and lock the crystal arm.
12. Click the Load Stage button to move the sample stage to the correct position to start your map. (The stage was centered and focused in previous steps.)
13. Collect a Spectrum background (rather than a Map background). This allows using the %T final format later in preview mode when monitoring contact. (This step is optional but recommended.)
14. Clear the mosaic to remove temporary frames, then draw the area map around the current centered position. The magnification of the germanium crystal is not indicated in coordinate values; they are 4x larger than the mapped area. The drawn map is typically larger than the live video pane. Ensure the map area is centered. Use the background point as a reference.

Maintenance

Follow these steps for operation and maintenance:

1. Open the crystal arm, then use the pressure column control knob to elevate the sample until it is visible in the video camera. Use the positioning screws on the accessory (instead of the joystick) to fine-tune the sample's position. Do not move the motorized stage.
2. Use the pressure column control knob to lower the sample until it is completely out of view. This should allow enough clearance for the crystal arm to close.
3. Close and lock the crystal arm.
4. Collect the background map.
5. Raise the sample and monitor the pressure indicator. The pressure reading will increase rapidly upon contact with the sample. Alternatively, activate preview mode and watch the spectrum as you raise the pressure column. When a spectrum appears, stop increasing pressure; further increases usually do not increase absorbance.
6. Collect the sample map.
7. Use Process > Map Math to ratio the sample map to the background map to create a reflectance map result.
8. Use Save Map As to store the ratioed map separately from the raw data, or Save Map to store the maps and overwrite the raw data.
9. If you are finished running samples for the day, install the storage disk. Refer to Installing the Storage Disk for instructions.

Contents

• Storing the Accessory
• Cleaning the Crystal
• Inspecting the Hydraulic Assembly
• Replacing the Crystal Arm and Crystal

Storing the Accessory

Before storing the accessory, ensure the red plastic storage disk is installed in the sampling area under positive pressure. Refer to Installing the Storage Disk for more information. The disk is required to maintain proper operation of the hydraulic assembly and protects the germanium crystal from being scratched by metal parts.

Cleaning the Crystal

Clean the crystal thoroughly before collecting background data and after measuring each sample. Use a clean, soft cloth, lens paper, or a cotton swab.

• NOTICE: Do not use laboratory wipes (too rough) or paper that is scented or contains lotions, as they will show up in your spectra.
• NOTICE: Use only water, alcohol, or hexane to clean the crystal. Other solvents may damage the crystal or other components.
• NOTICE: Cleaning fluid should be at or close to room temperature (or the temperature of the previous sample). Applying extremely hot or cold liquids may crack the crystal.

To clean the crystal

1. Dab the crystal with a clean cloth dampened with a recommended cleaning fluid, or place a drop of fluid on the crystal and wipe to remove most of the sample.
2. Repeat with a clean cloth.
3. If a solvent was used, allow enough time for it to evaporate completely or dry the crystal with clean, dry air or nitrogen.

Inspecting the Hydraulic Assembly

Keep all sharp sample preparation tools away from the accessory. Periodically inspect the tubing and tubing fittings for signs of abrasion or small cuts. If accidental damage is observed, contact your sales or service representative to order a replacement hydraulic assembly. If fluid leakage is observed, immediately stop using the accessory and remove it from your microscope.

Replacing the Crystal Arm and Crystal

If the crystal becomes damaged or broken, you can order a new crystal arm and replace it yourself. To order parts, contact your sales or service representative in your area or use the information at the beginning of this document to contact Thermo Fisher Scientific.

To replace the crystal arm

1. Use a thin spatula or screwdriver to remove the cap from the pivot screw.

2. Use a 3/32 inch hex driver to gently remove the hex head pivot screw, the crystal arm, and up to four washers above and below the arm. Your accessory will have one dome-shaped spring washer and between zero and three flat shim washers installed above and below the crystal arm. Look for shim washers that may have remained on the receiving post or are stuck to the bottom of the crystal arm bearing during disassembly.
3. Install the screw, spring washer (convex side up), new crystal arm, shims (if necessary), and cap according to the diagram in Figure 9. Use the same number of shim washers with your new crystal arm as were removed from the old one. The shims allow the arm to engage correctly with the arm lock support. The upper washer is a cone-shaped lock washer; the convex side must face up.

4. Use the hex driver to tighten the pivot screw until the arm swings freely but with some friction.
5. Replace the cap.

Appendix A - Total Force Applied by the Pressure Column

The pressure column has a natural spring rate that is overcome as the column advances. This makes the total force applied to the sample at a given internal pressure (shown on the integrated pressure indicator) dependent upon the actual thickness of the sample (including the spacer) mounted in the accessory. Figure 10 shows the typical behavior of the Imaging ATR accessory for 1, 3, and 5 mm thick samples at indicated internal pressures between zero and 40 psi.

Note: Avoid operating the accessory at internal pressures over 40 psi.

¹ Thickness values shown above include sample and spacer.