Installing and Using the Sable Systems Isotope Analyzer

The Sable Systems Isotope Analyzer is designed to be paired with the Promethion metabolic and behavioral phenotyping systems to enhance your exploratory power. This analyzer is complementary to traditional indirect calorimetry approaches and provides users with real-time measurements of how and when specific organic molecules are being oxidized to meet an animal’s metabolic demands.

When animals are given a dose of carbon-13 or oxygen-18 labeled tracer molecules, their bodies process that molecule at a rate that is specific to the physiology of each animal. They ultimately release carbon-13 dioxide or oxygen-18 carbon dioxide that can be measured in the breath. This analyzer measures the amounts of these isotopes in the breath every second and can be used to detect physiological differences caused by countless experimental interventions limited only by your imagination. In most cases, your isotope analyzer will be located on a cart and work alongside your Promethion core CGF system. However, this isotope analyzer can also be retrofitted for Sable customers that already have GA or FR type analyzers. Today I’ll show you how to use the Sable Systems carbon-13 and 18 oxygen isotope analyzer.

Let’s start with the plumbing. The newest CGF systems come equipped with two accessory analyzer ports on the front that allow additional gas analyzers, like the isotope analyzer, to subsample gas streams in parallel with your existing respirometry gas analyzer chains. Your support engineer can describe the specific sample locations for GA, FR analyzers, and other early model CGF analyzers. Note that a 16-cage system will ideally have two banks of analyzers, A and B, each one measuring eight multiplexed cages. On the other hand, a single isotope analyzer could also be used to measure either cages 1 through 8, bank A, or cages 9 through 16, bank B, as long as the gas inputs on the front of the CGF are placed in the correct position. The gas from the CGF inlet port is pulled, not pushed, through a braided Nafion segment to reduce the moisture in the gas sample to ambient levels. The gas stream then passes through an in-line 0.1-micron filter to remove any particulates. It’s best practice to replace this filter on an annual basis or as needed.

The gas stream then enters the laser cavity, where the magic happens. After the isotope analysis, the gas is pulled into a very powerful pump that maintains a strong vacuum inside the laser cavity. This pump should always be powered off if the isotope analyzer is not in use, otherwise damage to the pump could occur because the flow paths are closed. For the vacuum, I recommend using a mid-range setting on the duty cycle knob that’s located just above its power switch. The gas exiting the pump passes through an adjustable needle valve that’s manually set to a point where the downstream rotameter device reads a value of approximately 400 to 600 milliliters per minute. Lower flow rates are possible for special applications, but that requires some additional equipment. The rotameter has its own needle valve, but this should remain fully open to avoid creating unnecessary back pressure. Finally, the gas stream is vented back into the atmosphere.

Now let’s look at the wiring. The analyzer has a main power switch located near the gas sample intake and exit ports. If it is off, the power to the vacuum should also be switched off. At the other end of the analyzer, we see where the USB mouse can be plugged in. Note that all these USB ports are equivalent to one another. The VGA port is connected to a computer monitor that is supplied with your isotope analyzer. The mouse and the computer monitor are not always required for routine use with the Promethion system, but they are needed to conduct diagnostics and to change any settings within the isotope analyzer.

A null serial cable connects the serial output socket to the Promethion Data Hub, and this is a small black box about the size of a deck of cards. This Promethion Data Hub allows the real-time data from the analyzer to be streamed into your existing IM3 real-time software. The Promethion Data Hub then connects to a CAN port on your GA or CGF analyzer using a standard RJ45 cable. Note that no dedicated power input is required for this hub.

Let’s explore how the isotope analyzer communicates with the IM3 software. When your isotope analyzer is connected to the CGF and then powered on, your IM3 software will automatically detect the data streams. As you normally would, open up a web browser and then enter the IP address for your Promethion system. This gets you into the IM3 home screen. So far, everything looks good on this screen. Now click on the Status tab in the upper right corner of the screen. Then select the Gas Analyzers tab to view their status. Beneath the normal analyzer values, you’ll see a section for the isotope analyzer. In this case, we have the isotope analyzer connected to bank A, that’s cages 1-8. The values reported include the total CO2 concentration, the water vapor levels, the carbon-13 value, and the oxygen-18 value that are measured in the carbon dioxide of the gas stream. The vacuum pressure within the isotope analyzer is also shown, and this value shows you if your vacuum pump is on. Now if you have two isotope analyzers, they will both be visible on this status screen.

So go ahead and turn on the isotope analyzer using the switch located near the gas sample ports. During power-up, you’ll see a series of boot-up screens on the monitor. After a few seconds, you’ll be asked if you want to start the isotope analyzer. Click on this button to start the recordings. Now if you don’t click on this button, the isotope analyzer will automatically start itself within two minutes. Once the isotope analyzer is on, then you can turn on your vacuum pump. Note that any time the isotope analyzer is on, it’s recording data, even if the Promethion system is not actually connected to it. Later I’ll show you how you can access these data.

Now the default screen shows the laser spectra, and each of these peaks represents a specific gas species, and this screen is usually useful for diagnostic purposes only. Next, click the Display tab at the bottom left of the screen. The first screen you’ll see shows the real-time data updated at 1 Hz, or once per second. Now there are always two graphs available, and you can select which of the four possible gas species you want to be plotted on each one. Now even though only two variables are plotted, the bottom of the display screen shows the real-time values for all four gas species updated in real-time. As you can see, we’re now displaying the CO2 levels and the carbon-13 values in the ambient air. We now switch to a bottled calibration gas that has a higher CO2 content. Note that these graphs auto-scale themselves, and you can click the Refresh button to clear the visible history and allow rescaling of the plots for current data. Also note that the vacuum pressure of 120 Torr is shown at the bottom of the screen. This is a way of ensuring that the vacuum pressure inside the laser cavity is correct.

Now click the Display tab once more. This shows you the real-time variables in large font, and this can be useful if you’re trying to read the screen from across the room. Click the Display tab again. This page shows you the alarm statuses. All status indicators should normally be green, but they could be yellow or red if there’s a maintenance issue.

Now click the Setup tab on the bottom right side of the screen, and then select the Time/Files tab. The baud rate should be set at 115K for proper communication with the Promethion Data Hub. This is also the page where you can set the time zone, or you can synchronize the Isotope Analyzer clock with other computer clocks. When you’re finished here, click the Close button at the bottom of the screen.

Now move to the Calibration tab. I do not recommend adjusting any of these settings without external guidance. Now move to the Service tab on the far right. Following a calibration, the timer counter can be reset, and this will clear some of the alarm settings. The Laser Adjust, MIU, and the DCS screens are not to be used. Click the Close button to return to the home screens.

Now click on the Rate tab at the bottom of the home screen. You’ll see that the default frequency for data recording is one second, and this is a perfect choice for Promethion measurements because all the variables are measured at the same frequency. In special cases, perhaps if you’re making a very long recording using the Isotope Analyzer only, you may wish to change this setting to 10 or 20 seconds, so the resultant text files are not unmanageably large. Now click on the Cancel or Save button to exit this screen.

If you’re not using your Promethion system, you can still access your data using only the Isotope Analyzer. To do this, access the Files tab at the bottom of the screen. Now if you scroll down to the bottom of the list, you’ll find the most recent recordings that are made by the instrument. In normal use cases, your Promethion system will have already intercepted and logged the data that it needs. However, if a user wants to download an entire file, they’re all stored as compressed text files. To save one of these files, insert a USB drive into any empty USB port on the Isotope Analyzer. Then click the Mount USB button. Select the file that you want and then drag it onto the USB device. It’s important to click the Unmount USB button prior to removing the drive, otherwise the files may not be saved. Now click Close to exit this screen.

When you’re not using the Isotope Analyzer, power it off by clicking the Exit button. This ends the current recording, and now you can shut off the vacuum pump. Once the vacuum pump has powered down, you can switch off the main Isotope Analyzer.

In this final section, I’ll show you how to open a typical data file from the Isotope Analyzer. Note that if you’ve been using your Promethion system, you’ll never need to do this. First open your Excel program, then select Open New File. Now because you want to open a text file, it may not show up as an option. If that’s the case, change the file type to All Files. This will allow you to see the text file that you want to access. Once the text file is open, you’ll need to use the Text Wizard to import the file into Excel. Note that your data is delimited and has headers. The delimitation format is comma. Click Next and Finish, and you can see all your raw data. You may want to reduce the font and format the numbers so that they’re not in scientific notation.

Now I’ll show you some of the more interesting columns here. We’ll highlight CO2 in peach color. We’ll highlight the CO13 column in gray. The oxygen-18 values are highlighted in green. And the vacuum pressure is highlighted in blue. Finally, the water vapor column is highlighted in yellow. Now be sure to save this file as an Excel file, otherwise the changes that you make to this text file may not be saved. Good luck and happy experimenting!