Perfluorodecalin improves in-vivo confocal depth resolution in air-filled tissues

by  George Littlejohn & John Love

School of Biosciences, The University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter, EX4 4QD. U.K.

Laser scanning confocal microscopy (LSCM) enables optical sectioning of specimens. Criteria for confocal image acquisition are demanding to satisfy [1], particularly when imaging through several cell layers of differentially refractive tissue, as is the case for the spongy mesophyll of plant leaves.  The spongy mesophyll is located adjacent to the lower epidemis of leaves, contains numerous airspaces and may be several cells thick.  These characteristics result in high levels of light scattering within the mesophyll and a progressive attenuation of excitation laser intensity and decrease in emission intensity through the tissue, producing aberrations that impair confocal image quality [2]. We found that mounting leaves infiltrated with perfluorodecalin (PFD) significantly improves the resolution of confocal images of the mesophyll and enables imaging over two-fold deeper into the tissue compared to using water as a mounting medium [3].  The optical and physiochemical properties of PFD are particularly suitable for this application.  PFD is non-fluorescent across the excitation spectrum, from 200 nm to 700 nm [3], has a refractive index of 1.313 (, which is close to that of water (1.333) in which live specimens are commonly mounted and has a very low surface tension [4] which, unlike water [5], allows it to easily penetrate stomatal pores and fill the intercellular airspaces of the mesophyll; leaves kept in air or immersed in water are relatively opaque, whereas leaves bathed in PFD immediately become translucent (Figure 1) as the specimen becomes more optically homogenous.  We determined that PFD readily infiltrates the mesophyll without the application of a potentially destructive vacuum or wetting agent [3].
Perf fig1
Figure 1. Leaves incubated in air, water or PFD [3].
Protocol summary
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 ( 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 [6], which suggests that physiological stress is minimal [3]. 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 [7], the production of artificial blood substitutes [8] and lung inflation in premature babies [9]. PFD has also been used in the oxygenation of growth media, including those used for culturing plant cell protoplasts [10]. 
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.
perf in plants
Figure 2.  Shows confocal imaging of leaves from A. thaliana plants constitutively expressing cytoplasmically localised “Venus”, a variant of enhanced yellow fluorescent protein [12] mounted in air, water or PFD. Leaves mounted in air, typically show high levels of reflection from the surface of the epidermis that impairs image quality and results in poor Z-plane resolution.  Mounting leaves in water decreases reflections from the surface, enabling accurate imaging of the epidermis and of the mesophyll to a Z-plane located approximately 25 µm from the surface of the leaf.  Mounting leaves in PFD more than doubled the Z-plane resolution compared to water, allowing clear images to be acquired from 50 µm into the mesophyll.  Most importantly, this improvement in Z-plane resolution with PFD was achieved without increasing the power of the excitation laser, so reducing the potential for fluorophore bleaching and minimising cell damage.


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