The overall goal of this method is to quantify the spatial distribution of metals in tissue sections using laser ablation inductively coupled plasma mass spectrometry. This method can answer key questions of both basic biology and disease research, particularly for conditions where metal biochemistry is involved in disease pathology. The main advantage of this technique is it retains spatial information about metals which is traditionally lost using bog metal analysis techniques.
The implications of this technique extend toward therapy and diagnosis of neurodegenerative diseases because it allows us to monitor how metals change in brain regions that exhibit cell death. Generally, people new to this method can struggle, by setting up these long and complex experiments. Visual demonstration of this method is critical, as designing the experiment and analyzing data can be challenging because each step must be executed in a specific order.
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 ICPMS 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 two volume ablation cell every ten minutes move the sample stage to each corner of the chamber to reduce the amount of residual air in the cell.
Turn on the ICPMS 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 three 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 per 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 ICPMS 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 warmup 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 ICPMS 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 warmup and washout times.
Start the laser sequence, and check that the ICPMS correctly starts data collection after ablation. Once the data has been collected, extract the data for the standards from the CSV files. Perform baselines of traction, 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. The metal content and distribution of a wild type mouse brain was investigated using this technique.
Here, the distributions of carbon, magnesium, and phosphorus, are shown in counts per second. Quantitative images are shown for manganese, copper, iron, and zinc. The carbon distribution was used as an internal standard.
Iron was most abundant in the mid brain and the dentate gyrus. Zinc was most abundant in the cortical areas. The information from these images can then be used to track the expression and functionality of metal binding proteins Once mastered, an experiment spanning for several days can be set up within an hour when done properly.
While attempting this procedure, it's important to synchronize your ablation lines and your data files with ICPMS. Following this procedure, additional methods like histological staining on adjacent sections can be performed in order to provide additional information like the colocalization of metals with specific proteins. We first had the idea for this method when we wanted to examine how metal levels changed in laboratory models in Parkinson's disease.
Though this method can be used for gaining insight into neurodegeneration it can also be used for different tissue types and disease states, including human tissue, and a range of biological matrices. After its development, this technique paved the way for researchers in the field of neuroscience to examine how metal levels changed in both human samples and animal models of disease. After watching this video, you should have a pretty good idea of how to set up the laser ablation ICPMS experiment.
Don't forget that working with biological material can be extremely hazardous and precautions such as wearing appropriate personal protective equipment should always be taken while performing this procedure.
