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

Place a mouse brain tissue sample and standards in a laser ablation chamber.

The standards contain known concentrations of metals spiked into lamb brain tissue homogenates.

Fill the chamber with argon gas and focus an ultraviolet laser beam on the tissue.

The laser vaporizes the sample and standards in a linear pattern, creating an aerosol of fine particles.

Argon gas carries the particles into the inductively coupled plasma, a high-temperature ionization source.

The plasma atomizes and ionizes the particles into positively charged ions.

The ions enter the mass spectrometer and are separated based on their mass-to-charge ratios.

The resulting data are compared to a reference database to identify the metals present in the sample. From the standards, a calibration curve is generated.

Calculate the metal concentrations in the sample by calibrating its signal intensities against those of the standards, revealing the spatial distribution of metals within the brain slice.

First, place a brain tissue sample and all matrix matched tissue standards in the laser ablation chamber. Ensure that the samples are within the LACCD camera depth of field. Close the chamber door and finger tighten the screws.

Then in the ICP-MS software, open the carrier gas valve and set the flow rate to 1.2 liters per minute. In the LA software, run a 30 minute purge of the ablation cell with the carrier gas for a 2 volume ablation cell. Every 10 minutes, move the sample stage to each corner of the chamber to reduce the amount of residual air in the cell.

Turn on the ICP-MS and allow the instrument to warm up for two hours. While the instrument is warming up, in the laser ablation software, draw a line of ablation approximately 3 millimeters long across the surface of the first tissue standard. Click the line of ablation and set the default beam diameter, the scan speed, and the radiant fluence. Duplicate the line six times, spacing the lines apart by the beam diameter. Repeat this process for each standard.

Then, use the Line tool to draw the ablation area over the sample from the upper left corner across the widest point of the sample, using the same parameters as for the standards.

Duplicate the line as needed for complete coverage of the sample with the same spacing as used in the standards. Then draw a set of lines for intermittent scanning of standards no later than after 20 hours of scanning the sample or between each sample. Add a scan of the standards to the end of the experiment.

Based on the line length and the scan speed, determine the analysis time for the sample and standard lines. Then create a new batch in the ICP-MS software and select Time Resolved Analysis. Choose the mass to charge ratio values of interest, and set the integration times for each ratio, so that the total integration time for one cycle is 0.25 seconds. Save the batch.

For a standard batch, fill in the analysis time for each line with an additional 15 second buffer to account for laser warm up and washout times. Set a sample run list with an acquisition for each standard line. For the sample batch, duplicate the standard batch and adjust the times and number of acquisitions accordingly for the sample ablation pattern.

Once the ICP-MS has warmed up, add the first batch to the queue and ensure that the software is waiting for a trigger from the LA instrument. Then, in the LA software, turn on the laser power supply and set the laser warm up and washout times. Start the laser sequence, and check that the ICP-MS correctly starts data collection after ablation.

Once the data has been collected, extract the data for the standards from the CSV files. Perform baseline subtraction and exclude the background signal. Identify signal drops caused by holes in the tissue standards.

Mask these low signal areas and then export the data as a spreadsheet. Calculate a conversion factor from CPS to PPM for each element identified. Repeat this process for each standard.

Next, open the quantitative image construction software and import the sample data from the CSV files. Select several areas of the background to apply background correction. Select the graph editing tool, right click the image, and select Modify Image Appearance. Change first color at Z to a large negative value.

And click Done to enhance the background signal. Then in the Standards tab, enter the CPS PPM correction factors for each standard and click Go. Open the Data Browser in the Data tab, right click on a corrected image file, and click New Image.

Open the Modify Image Appearance window and set the color table and scale. Use the Add Annotation utility to place a color scale on the image. Adjust the color scale in the color scale window. Once the image has been colored and annotated, either copy and paste the image into another program or export the image from the image construction software.

To activate the ROI tools, select Image Processing from the Analysis packages and choose an image. In the Image tab, click ROI and start ROI Draw. Select the region of interest and then click Finish ROI.

In the Image tab, click Stats to obtain the ROI data. Copy and paste the data to a spreadsheet, making sure to save when each ROI is added. Repeat the process as needed.