Single-Cell Free Radical Measurements
What is T1 Relaxometry
T1 Relaxometry in Quantum Sensing is a technique that utilizes the behavior of electrons in a nitrogen-vacancy (NV) center in a nanodiamond. A carbon atom is replaced with a larger nitrogen atom, which causes a vacancy to form next to the nitrogen, creating an NV center. The electrons in an NV center are sensitive to magnetic interference and alter their behaviour in terms of fluorescence. The electrons can be excited with lasers in the range of 500-600nm. The excited electrons will emit photons in the range of 650-800nm. We excite the electrons with varying intervals between a laser pulse. By altering the length in the pulses, the excited electrons fall back into the ground state at different rates depending on the magnetic interference. If we plot the initial re-excitation after a dark time versus the dark time, we create a T1 relaxation curve. By analyzing the curve, a T1 relaxation time can be extrapolated. The T1 curve changes significantly depending on the presence of magnetic interference from free radicals.
Relaxometry in Cells
Nanodiamonds can be used to detect a large variety of samples. From materials to currents and free radicals in cells. At QT Sense we focus largely on the latter. Nanodiamonds can be on the surface of the cells or inside the cells via endocytosis. Nanodiamonds can be directed to specific organelles or just the general cytoplasm. The nanodiamonds can be measured with specialized equipment, the Quantum Nuova, to get a measurement of free radicals around the nanodiamond. Effects of drugs, stressors or any other effect on cells can be monitored.
Figure 1: Typical workflow to measure free radicals in single-cells. Nanodiamonds are added to the cells, they are incubated over time. The cells are placed in the Quantum Nuova and the results are displayed.
Operation of the Quantum Nuova
The Quantum Nuova is a modified confocal system which is especially suited for T1 relaxometry measurements. The sample, whether liquids (such as blood), cells, or tissues, is grown under normal conditions following standard protocols.
1. The sample container is placed in the machine on the objective.
2. The software designed uniquely for the Quantum Nuova, helps with finding the appropriate nanodiamonds. The nanodiamonds need to be within a certain intensity, size and placement. If for a specific experiment, the nanodiamond is required to be at a specific organelle, this can be verified.
3. The nanodiamond is tested with regards to photobleaching (nanodiamonds do not photobleach, allowing prolonged experiments)
4. In the software, Quantum Pulse, all the parameters for the T1 experiment can be controlled (the dark times between pulses, step size, etc.).
5. The Quantum Nuova will then initiate the experiment and provide a fully automized analysis of the data. The T1 relaxation curve and time will be displayed on the screen.
The experiments depend on the selection of appropriate nanodiamonds and the correct T1 parameters. At QT Sense we can help with determining the best settings for your experiments and the software selects the most suitable nanodiamonds to make your experiments as successful as possible.
