The overall goal of this procedure is to minimize degradation of biological images caused by the airspaces in plant leaves to acquire clearer images of living cells within the leaf. If the leaf is mounted in an aqueous mounting medium and a fluorophore is excited by a laser, many unfilled air spaces and changes in refractive index within the tissue shown in orange limit the strength of the signal deep from within the tissue in the resultant image. If the leaf is instead mounted in per fluoro deline or PFD, the resulting laser excitation of the fluorophore is less attenuated and allows better transmission of emitted signals.
Ultimately, use of scanning laser confocal microscopy with the PFD mounting medium gives clear images of living cells within the plant leaf at a depth that is more than double that possible when an aqueous mounting medium is used. The main advantage of this technique over existing methods like fixation or mounting leaves and aqueous media, is that it reduces refraction within the meso fill and enables more accurate imaging of living cells deeper within the leaf, which has been headed to problematic. This method can help answer key questions in plant science, such as addressing meso fill, cell biology, and signaling in vivo, the colonization of leaves by pathogens and pathogen fill Interactions, Though this method can provide insight into fundamental plant biology.
It can also be applied to other highly refractive tissues that are rich in airspace such as insight spirals or the vertebrate lung. Demonstrating this procedure will be Dr.George Littlejohn from my laboratory To Mount Leaf. Samples in PFD begin by preparing a microscope slide with a gas permeable gasket of poly dimethyl Sloane or P-D-M-S-P-D-M-S is a viscoelastic polymer that can be molded to provide a chamber tailored to experimental requirements.
Equilibrate the PFD with air by shaking a small volume of PFD in an air-filled bottle. This may also be achieved by bubbling air through the PFD next, decant the PFD into an open Petri dish and float a whole seedling or excise leaf on the liquid for five minutes. The tissue should become translucent reminiscent of vitrified tissue.
Leaves may appear darker or lighter than before exposure to PFD, depending on the lighting conditions and the age of the tissue used. Finally, fill the PDMS chamber with aero equated PFD and carefully transfer the tissue samples from the incubation Petri dish to the PDMS chamber. Seal the slide with a cover, slip and image.
According to experimental requirements, months leaves were incubated for five minutes in a suspension of recombinant green fluorescent protein or GFP. In PFD microscopic inspection of PFD incubated leaf samples shows that the majority of air spaces are flooded with recombinant GFP suspended in PFD. The GFP is in green, and the red represents chlorophyl, lotto fluorescence and delits meso fill cells.
It is apparent that the leaf is flooded upon incubation with PFD, but that occasional pockets of air may remain Water does not flood the leaf under these conditions. Here, a opsys leaves expressing Venus, a variant of YFP, which localizes to the cytosol are incubated and mounted in air, water or PFD laser scanning. Confocal microscopy of the samples shows an advantage of PFD over air and water with an approximate twofold increase in depth of imaging.
When comparing PFD and water Once mastered, this technique can be done in five to 10 minutes if performed properly. While performing this procedure, it's important to remember that peripheral delin dissolves nail varnish and Teflon, but it's compatible with silicone grease. After watching this video, you should have a good understanding of how to use per fluro delin as a mounting medium to improve imaging in cells of plant leaves.
Don't forget that while per fluoro delin is non-toxic, we recommend that standard safety practices be adopted when performing this procedure.
