The NightShade In Vivo Imaging System for plants is a very flexible instrument, and its range of applications is really broad, but we are going to focus on some of the most frequently used applications, plus some less frequent but also very interesting ones, namely:
Split luciferase complementation is a popular method to study protein-protein interactionsin planta by using an in vivo imaging system to visualize luminescence on transformed leaves. It is based on the complementation of two cleaved fragments of a luciferase protein and its subsequent detection by substrate conversion and light production. The reconstitution of luciferase is reversible in most cases, and therefore suitable for monitoring dynamic interactions in real time. Both Firefly luciferase and Renilla luciferase have been successfully used to study PPI in vivo.
The Nightshade is a very popular instrument for this method thanks to of its highly sentitive camera and flexible software.
Picture reproduced from "Jing, Y., Liu, J., Liu, P. et al. Sci Rep 9, 5691 (2019). https://www.doi.org/10.1038/s41598-019-42177-y" under the Creative Commons Attribution 4.0 International Public License.
Monitoring the expression of genes involved in circadian rhythms often requires imaging during many hours and in controlled light conditions. The NightShade is the ideal tool for this application, thanks to its unique design:
- Daylight simulation with flexible light quality and intensity.
- Intuitive software with powerful scheduler, which allows to program the desired intensity and quality of light during several days and capture images at the desired time points.
- Light-tight ports to insert tubing for irrigation and other goals.
- Side camera mounting position to image whole plants, from leaf to roots.
- Anti-condensation tray with temperature control for high quality images when working with petri dishes.
Monitoring stress in plants using in vivo imaging can be performed in wild type, unmodified plants, thanks to two important properties:
Delayed fluorescence, also called afterglow, is the weak light emitted by pre-illuminated intact plants. It acts as an indicator not only for chlorophyll content, but also for the physiological state of the plant which can vary based on environmental influences such as drought, high saline levels or infections. The signal of prompt fluorescence lasts for nanoseconds whereas delayed fluorescence can be detected seconds and minutes later. In the two pictures at the top you can see the difference in delayed fluorescence between a well irrigated plant (left) and the same plant after 2 days of drought (right).
In addition, in response to stress such as wounding, salt stress or pathogen attacks, an ultraweak biophoton emission is generated. Its intensity is usually in a range less than 1000 photons/s*cm and correlates with the second burst of ROS, but it is independent of it. The molecular mechanism underlying this light emission is unknown, but it offers a nondestructive and facile method to investigate processes related to pathogens and other stress factors. Biophoton emission can last for hours, and biophoton imaging can thus be performed once delayed fluorescence has faded away. In the bottom picture you can see the biophoton emission in response to increasing concentrations of cadmium. Picture reproduced from Jócsák, I., Malgwi, I., Rabnecz, G., Szegő, A., Varga-Visi, É., Végvári, G., Pónya, Z., 2020. PLOS ONE 15, e0240470. doi:10.1371/journal.pone.0240470 under the Creative Commons Attribution 4.0 International Public License.
Pathogens of interest can be genetically engineered to express bioluminescent or fluorescent proteins. This allows to monitor how an infection progresses in time, space, and in response to several factors.
The picture displays a very nice example of this application: bacterial infection dynamics can be perfectly visualized in real-time in reciprocal grafts of a resistant (BVRC 1) and a susceptible (BVRC 25) line after root-dip inoculation with the bioluminescent R. solanacearum strain BL-Rs7. Reproduced from Du, H.; Wen, C.; Zhang, X.; Xu, X.; Yang, J.; Chen, B.; Geng, S.. Int. J. Mol. Sci. 2019, 20, 5887. doi.org/10.3390/ijms20235887.
The NightShade is very well suited for this application, thanks to its powerful scheduler software and high sensitivity, which allows to visualize the pathogen even at early stages of the infection.
And there are many other possible applications of the NightShade:
- Of course, visualizing gene expression patterns. The unique side camera position of the NightShade makes it possible to image the whole plant, from root to leaves, without problems caused by geotropism in long experiments.
- Gene expression screening is also a popular application: different sample holders are available for up to 9 petri dishes, allowing to screen many seedlings in a short time. The picture on top displays an example of a mutant screening (Reproduced from "Thatcher, L.F., Foley, R., Casarotto, H.J. et al. Sci Rep 8, 13454 (2018). https://doi.org/10.1038/s41598-018-31837-0" under the Creative Commons Attribution 4.0 International Public License).
- The customizable multiport flange opens up a world of possibilities: do you need to irrigate the plants inside the dark chamber? No problem. Do you need light guides for custom lightning? Easy. Do you need to touch the plants to study touch-sensitive genes? Why not!
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