The overall goal of this procedure is to acquire long-term observations of a plant root system using light sheet microscopy. To achieve this, it is essential to have a gentle sample preparation method, controlled growing conditions, and an imaging technique that does not alter plant development, such as light sheet microscopy. To begin, plants are grown on the surface of a two-millimeter thin layer of gel.
The gel with an Aribidopsis seedling is then cut out, scooped up and transferred onto the microscope sample holder. Inside the microscope, the plant grows in its natural upright position. The roots grow in a liquid medium, while the leaves remain in the air.
A lighting system illuminates only the leaves. The water surface is covered in order to keep the roots in darkness. One of the main advantages of this technique over existing methods, such as embedding the root inside of the gel is that we are used to grow plants on the surface of a gel.
Therefore, we can stick to the very same growing protocol as before. And second, the root is in direct contact with the liquid medium, which can be exchanged for drug treatments, for example. Pour 30 milliliters of freshly autoclaved half-strength MS medium containing 1.5 percent phytogel in a square petri dish to create a layer of about 2 millimeters.
Sow sterile seeds side by side with at least one centimeter space in between. Then, wrap the plate with a micro-pore tape. Cultivate the plate in your growth incubator vertically for up to 10 days.
In this experiment, we use six-day-old plants to observe the development of lateral roots. We provide a model for 3D printing the sample holder. We have tested different materials.
The transparent resins were most suitable in terms of rigidity and water resistance. A detailed description of the dimensions to machine a sample holder from solid material are described in the manuscript. Cut the gel around the plant using a scalpel.
Then, scoop up the block of gel on which the plant is growing on and trnasfer it carefully to the sample holder. Melt 1 percent aggros at 80 degrees Celsius and cool it down to 33 degrees Celsius. Glue the block of gel onto the sample holder and glue the plant on the gel carefully.
Use a stereo microscope to verify the position of the plant and make sure that the region of interest is not covered by any gel. In order to prevent the plant from drying out, slide the sample holder onto a 1, 000 microliter pipette tip whenever possible. Put the holder onto a pipette box and prepare more samples if required.
Our microscope is a classic open-spin with no major modifications. We implemented a lighting system for culturing plants inside the microscope. Red and blue LEDs are arranged in a ring.
Pairs of LEDs can be switched on and off individually for directional lighting. The spectrum of the lighting system matches the one in our growth chambers. Almost all of the lighting coming from the LEDs is blocked by emission filter for GFP.
We could not detect any stray light reaching the camera. Hence, the light can be kept on while imaging. The light intensity can be adjusted, ranging from 30 to 250 micro moles per meter squared per second.
Screw or glue the LED ring on the lower side of the open spin stage arm. Mount it back to the microscope and hook up the wires. Assemble the profusion system.
Connect the tubes to the sample chamber in a one-way arrangement. Adjust the speed to one milliliter per minute. Add three millimeter squares of a sterile black plastic foil and distribute them on the water's surface.
Insert the sample into the microscope. Verify that there are no plastic sheets sticking to the sample holder. Close the chamber with two lids made of black aluminum foil.
Turn on the light and adjust the light intensity. Turn off the room light and start recording. This method allows to record root growth over several hours up to days, presented here by the growth of a lateral root.
Images of a z-stack of a single time point show good optical sectional properties. We captured the entire process of lateral root formation in 3D, which allows us to study the dynamics of this process. Looking at a single slice of this recording shows good resolution, even deep inside the root.
After watching this video, you should have a clear understanding of the plant sample preparation method for light sheet microscopy. Following this protocol, we now are able to look at intracellular protein localization or gene expression patterns during organo-genesis, plant tropisms or plant adaptation. This will help us to understand the spatial-temporal regulation of plant development.
