Figure 1. Leaves incubated in air, water or PFD .
Tissue preparation is easy, but should be tailored to suit the requirements of individual experiments and imaging procedures:
1) Float leaves or seedlings on PFD (Fluoros / F2 Chemicals (http://www.fluoros.co.uk/contact_us.php)) for 5 minutes. You should see the tissue darken and become translucent instantaneously as it is placed on PFD.
2) mount on a slide. We use a sealed chamber made with Carolina observation gel (Blades Biological Catalogue number 13-2700) to minimise evaporation of PFD.
Airspaces of the mesophyll are essential for gaseous exchange and we were concerned that completely infiltrating the mesophyll with PFD might, while improving image resolution, have a deleterious effect on the physiology of the plant and negate any experimental advantage over using fixed samples, so we assessed the physiological impact of PFD on plants. We monitored germination and growth of seedlings and measured Fv / Fm, an indicator of stress in photosynthetic tissues , which suggests that physiological stress is minimal . We suggest this may be explained by the exceptional O2 and CO2 carrying capacities of PFD that readily permit gas exchange between tissues immersed in PFD and the medium. This property has been exploited in several medical applications, notably in eye surgery , the production of artificial blood substitutes  and lung inflation in premature babies . PFD has also been used in the oxygenation of growth media, including those used for culturing plant cell protoplasts .
The use of PFD in multi-photon microscopy may further increase the depth penetration of that technique [2, 11]. Moreover, the properties that PFD has displayed for mesophyll, namely easy infiltration into the tissue, significant improvement in Z-plane resolution and non-toxicity, may be exploited for in vivo imaging of air-filled spaces, where gaseous exchange is also important and which are a primary target for microbial infection. Using PFD to improve the resolution of LSCM images will refine our understanding of the process of microbial invasion and survival within these critical tissues and potentially lead to advances in the treatment of crop diseases, and improved biocontrol methods.
In summary, PFD has significant advantages as a mounting medium for in vivo LSCM, most notably the increase in Z-plane resolution without a concomitant increase in excitation intensity that may damage cells. PFD is non-fluorescent, readily applied, and has minimal physiological impact on the mounted specimen.
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