Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry to Visualize Metals in Mouse Brain Tissue

Abstract

Source: Hare, D. J., et.al. Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS). J. Vis. Exp. (2017)

This video demonstrates the use of laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) to visualize and quantify metal distributions in mouse brain tissue. The protocol involves laser ablation to generate an aerosol of sample particles, ionization of the particles in high-temperature plasma, and mass spectrometry to separate the ions by their mass-to-charge ratios. Specialized software is then used to generate quantitative images, revealing the metals' spatial distribution within the tissue.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation of LA-ICP-MS for Analysis

1. Place standards and sample in the ablation chamber, ensuring that they are within the depth of field of the CCD camera fitted to the LA unit. If tuning the instrument is necessary, include a suitable reference material (e.g., NIST 612 Trace Elements in Glass). Finger-tighten the two screws on the chamber door to seal the ablation chamber.
2. In the ICP-MS software, select open the argon gas valve in the 'Maintenance' or similar panel and set the carrier gas flow to 1.2 L min^-1 in the appropriate dialogue box.
   NOTE: In this protocol argon is used as the carrier gas. Several examples use either helium or a mixture of helium and argon as carrier gas.
3. In the LA software, click the 'purge' button to flush the cell with argon gas for a minimum of 30 min.
   NOTE: Purge time can be altered by clicking a 'purge time' or similar button. When using an ablation system with a two-volume ablation cell, periodically move the stage to each corner and diagonally traverse the cell to ensure as much residual air is removed from the cell as possible. This can be achieved by selecting the 'home stages' or equivalent function.
4. Turn on the ICP-MS by clicking 'plasma on' and allow to warm up for two h, during which steps 1.5 - 2.4 can be carried out. Instrument settings vary between manufacturers.
5. Representative ablation of tissue standards
   1. Select the line tool and draw a single line of ablation approximately 3 mm long across the tissue surface.
   2. Set the parameters as follows by right-clicking the line of ablation in the experiment list and changing the following: beam diameter (selected as appropriate by the user; a 30 µm square beam is used here), scan speed (4 times the beam diameter per second; 120 µm s^-1) and energy fluence (0.3 -0.5 J cm^-2 for soft tissue; optimize if necessary for harder matrices). At 30 µm tissue thickness the laser beam does not penetrate the full thickness of this tissue, eliminating any potential contaminant from the microscope support. Normalizing to carbon can be used to correct for variation in the amount of tissue ablated. Set these parameters as the default for each subsequent line by selecting the 'default' radio button.
   3. Duplicate this line six times by selecting the initial line, right-clicking and selecting 'duplicate scans'. Make sure lines are offset in either the x- or y-axis by the beam diameter. This gives a total of seven lines per standard, spaced apart according to the beam diameter.
   4. Repeat steps 1.5.1 - 1.5.3 for each standard, ensuring the line of identical length.
6. To draw the ablation area over the sample, follow one of the two following methods:
   NOTE: Ensure the same scan parameters (beam diameter, scan speed, energy fluence) are used for sample lines.
   1. If the LA system is equipped with a wide field of view, select the line tool and draw a line from the top left corner of the sample long enough to cover the sample at its widest point using the same laser parameters outlined in 1.5. Duplicate this scan as outlined in 1.5.3 with lines spaced according to the beam diameter as many times as necessary to ensure complete coverage of the sample.
   2. If a wide field view is not available, determine and record the x and y coordinates (typically shown on the main screen as 'stage position' or similar) corresponding to the corners of a rectangle covering the entire sample. Use these coordinates to position parallel lines of ablation covering the entire sample area.
   3. Draw lines for intermittent scanning of standards at the latest after ~20 h of scanning the sample by repeating the process described in 1.5. Depending on the scan duration of the sample this may be required multiple times. End the experiment with an additional scan of the standards.
      NOTE: When determining the x and y coordinates while the cell is being purged, selecting home positions and the associated calibration of the axis will change the previously acquired specifics for the x and y coordinates whereas the difference (i.e. the length of the line) will be unaffected.

2. Setting Up Data Acquisition Methods for the ICP-MS

1. For the standard line of ablation, divide the length of the line by the laser scan speed to determine the total analysis time for a single line. Repeat this for the sample line.
2. In the ICP-MS software, create a new method (shown here as a 'batch') and ensure that 'time resolved analysis' or equivalent is selected. Select the m/z values to be detected, and then adjust the integration time for each m/z so the total integration time for one cycle equals 0.25 s. Click 'Save Batch As' and name accordingly (e.g., Std1).
   NOTE: As the sample will traverse the laser beam at four times the beam diameter, this ensures a data point for each mass is recorded equivalent to the beam diameter. For instance, using a 100 µm spot size, a scan speed of 400 µm s^-1 with an integration time of 0.25 s will produce images with true pixel sizes. Integration time can be adjusted to improve sensitivity; when increasing integration time to 0.33 s scan speed should be slowed to three times the beam diameter.
3. For standards, enter the analysis time for each line scan in the appropriate box, plus an additional 15 s to account for laser warm-up and washout times. Enter a sample run list (i.e. the order in which scans are run) with the same number of acquisitions (typically numbered sequentially; i.e. 001, 002, etc.) as the total number of standard lines.
4. For the sample, duplicate the method used for the standards by saving the current method or batch with an alternative file name, and adjust the total acquisition time (including the additional 15 s) and total number of acquisitions to match the number of lines that will ablate the sample.
   NOTE: As most LA-ICP-MS systems use a one-way trigger (LA triggers ICP-MS), it is essential that the ICP-MS software is awaiting the trigger from the LA before the subsequent line of ablation commences. The ICP-MS acquisition window will read 'waiting for start'.

3. Running the Experiment

1. Start the ICP-MS queue by adding the first method or batch to the queue and ensure the software is awaiting the trigger from the LA system.
2. In the LA software, enable the laser power supply by clicking 'emission', click 'run' and set the laser warmup time to 10 s and washout time to 20 s in the appropriate boxes.
   NOTE: This overrun will ensure the ICP-MS is ready to start acquiring new data when the laser commences each subsequent line of ablation.
3. Start the laser sequence by clicking 'start'. If using a two-volume cell, ensure the sample cup is in position.

4. Calculating Quantitation Standards

NOTE: There are multiple variations for converting ICP-MS data into images. These include the use of home-made software tools written in open-source languages, commercial macros and data analysis software. Here, use the recently developed software plugin, based on a specialized LA-ICP-MS data analysis suite.

1. Transfer all the batch folders containing the run data (001.d, 002.d, etc.) onto a separate computer with the analysis software installed. Extract the *.csv data files for each line of the batch into a separate folder.
   NOTE: Using a script to automatically transfer *.csv files into a new folder is highly recommended. See attached Python code for more details. This script has been written to suit either an Agilent 7700 or 8800 Series ICP-MS but can be edited to suit the output file from other manufacturers.
2. Open the software platform and follow the tabs sequentially to import and analyze the standards to produce quantitative images as described below.
3. To import the data from the first standard batch, select 'Agilent .csv' for 'File Type', 'Entire folder' for 'Import type' and check that the 'Date format' is identical with the computer format. Click 'Import', select the folder containing the .csv-files for the first set of standards, and move to the 'Baselines' tab.
4. Baseline subtraction
   1. Use the 'Automatic Selections' tab (command 2) from the main application dropdown menu in the toolbar, select 'Information from import' and click 'Continue'.
   2. Click 'Select All' and select 'Baseline_1' for integration.
   3. To select the first 10 s of each scan line (corresponding to the laser warm-up time), crop the data by entering second values '0' and '(duration of line - 10 s)' and click 'Add integrations' (e.g., for a 35 s scan line, enter values '0' and '25').
5. To select the sample data, use the 'Automatic Selections' tab as described in 4.4, but choose 'Output_1' as the integration. Crop data from the beginning and end of each standard line to exclude the background signal (e.g. for a 35 s scan line, enter values '13' and '4').
6. To exclude drops in the signal caused by possible holes in the standards, in the 'Samples' tab, click 'Channels' on the top left of graph area to select a channel with a high signal to noise ratio (e.g., C13 or P31). Make a note of any sharp drops in the signal and select a CPS value low enough that is below the normal variation within the samples, but high enough to pick out the sharp drops in the signal.
7. Click the 'DRS' tab and select 'Baseline_Subtract' for the 'Current Data Reduction Scheme'. Choose the channel from 4.6 for 'Index Channel' and the CPS value for 'Threshold to use when masking low signals'. Insert a value <0.5 s for 'Seconds to trim before/after low signals' to account for the delay in signal transfer from the laser to the ICP-MS.
8. To confirm adequate masking of the low signal areas, click back to the 'Samples' tab and select a processed (e.g., Fe56_CPS) channel trace. Repeat and refine the CPS and 'Seconds to trim before/after low signals' values as in 4.7 if necessary.
9. Click the 'Results' tab and choose 'Export data' from the main application dropdown menu. Enter a file name to generate a spreadsheet data file of the results (e.g., Std1 calculations, Std2…).
10. Open the data file in a suitable spreadsheet program and calculate the CPS values for each individual channel to be quantitated. Use the predetermined metal concentrations from the solution nebulization ICP-MS to calculate the conversion factor from CPS values to ppm (µg g⁻¹) for each quantifiable element.
11. Repeat steps 4.3 - 4.10 for all the sets of standards in the run.

5. Constructing Quantitative Images

1. Write 'Biolite()' in the command prompt to open up the software.
2. Import the sample data by clicking 'Load Images' and selecting the folder containing the .csv-files for the sample image.
3. Background correction
   1. Click the 'Baselines' tab to apply background correction for the sample. On the phosphorus (P31) image prompted on the screen, select areas of the background using the rectangle draw tool. Selecting as many regions as possible creates an image-wide map of signal/plasma drift to ensure adequate compensation from these confounding factors.
   2. To make the background signal more visible, select the graph edit tool (top left of the displayed image) and right click the image. Select 'Modify Image Appearance' and change the 'First Color at Z=' to a large negative value (e.g. -100,000). Proceed by clicking 'Done'.
4. Click 'Standards' tab to bring up a table for CPS/ppm correction factors. Enter values calculated from the standards in step 4.10 for each of the elements. This step will help to correct for the sensitivity drift in the ICP-MS. Click 'Go!'
5. Open 'Data Browser' from the 'Data' tab, which holds the images for each element and each step.
6. To finalize an image for export, select the desired image in the 'StdCorrImages' folder, right-click the image name (*_ppm) and click 'NewImage'. Right-click the image to open 'Modify Image Appearance' to choose a desired color table and color scale. Add a color scale to the image by right-clicking the image and selecting 'Add Annotation'. Select 'ColorScale' from the top-left dropdown menu and use the tabs to modify the desired color scale.
7. To export an image, make sure that the image in question is selected and navigate to the 'File' tab and click 'Save graphics…'. Select the desired format and save the image. Alternatively, the selected image can be transferred using the copy and paste tools.
8. Repeat steps 5.6 and 5.7 for all the images of interest.
9. Quantifying discrete regions
   1. To activate the ROI tools, navigate to 'Analysis' tab, 'Packages' and select 'Image Processing'.
   2. Select the desired image and navigate to the 'Image' tab and click 'ROI…'. Click 'Start ROI draw' and use a drawing tool to select a region of interest in the sample. To finish drawing, click 'Finish ROI'.
   3. To acquire the statistics for the selected ROI, navigate to 'Image' tab and click 'Stats…'.
   4. Copy and paste the results to a separate spreadsheet. Repeat ROI selection (5.9.2) for all regions and elements of interest.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Soda glass microscope slides	n/a	n/a	Typical slides are suitable for all experiments
NWR213 Laser Ablation system	ESI Ltd	n/a	Used in these experiments. Other manufacturers suitable, may require modifications to protocol
Agilent 8800 Series ICP-MS	Agilent Technologies	n/a	Used in these experiments. Other manufacturers suitable, may require modifications to protocol
Iolite	Iolite Software	n/a	Available from http://iolite-software.com/. Other methods are available, see protocol
Excel	Microsoft	n/a
IGOR Pro	Wave Metrics	n/a	Avalable from https://www.wavemetrics.com/products/igorpro/igorpro.htm