Name: A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium (Video) | JoVE Journal
Uploaded: 2012-01-16T13:00:00.000+00:00
Duration: 5 min 19 s
Description: Learn to image Arabidopsis thaliana leaves using perfluorodecalin as an infiltrative mounting medium. This method doubles imaging depth compared to aqueous media while preserving living cell physiology for plant biology research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors would like to thank Prof. Nicholas Smirnoff of the University of Exeter for his advice and the University of Exeter Bioimaging Facility. Funding was provided by the UK Biotechnology and Biological Sciences Research Council (grant reference BB/E002682/1).

The protocol given below describes a simple method for using PFD as an infiltrative mounting medium in Arabidopsis thaliana leaves, but we anticipate that this method may be used in a variety of applications where imaging air-rich tissues is desired.
1. Mounting leaf samples in PFD
<ol>
 <li>Prepare a microscope slide with a gas-permeable gasket of polydimethylsiloxane (PDMS, Carolina Observation Gel). PDMS is a viscoelastic polymer and can be molded to provide a chamber tailored to experimental requirements.</li>
 <li>Equilibrate PFD with air. This may be achieved by bubbling with air or by shaking a small volume of PFD in an air-filled bottle.</li>
 <li>Decant PFD into an open Petri dish and float a whole seedling or excised leaf on PFD for 5 minutes. The tissue should become translucent, reminiscent of vitrified tissue (Figure 2 (a)). Leaves may appear darker or lighter than before exposure to PFD, depending on lighting conditions and the age of tissue used.</li>
 <li>Fill the PDMS chamber with air-equilibrated PFD and carefully transfer the tissue samples from the incubation Petri dish to the PDMS chamber. Seal the slide with a coverslip and image according to experimental requirements.</li>
 <li>Note: PFD also performs well in an open chamber constructed on a coverslip or in a perfusion chamber and is compatible with silicon grease. PFD is not compatible with Teflon components as it dissolves them.</li>
</ol>
2. Representative Results:
Microscopic inspection of PFD-incubated leaf samples shows that the majority of airspaces are flooded. Figure 2 (b) shows airspaces flooded with recombinant GFP suspended in PFD. 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. LSCM of samples incubated and mounted in air, water or PFD shows that PFD gives an advantage over water and air in the depth of imaging possible (Figure 3). The example given in Figure 3 shows air, water and PFD mounted Arabidopsis leaves expressing cytosolically localized Venus, a variant of YFP11 that is expressed constitutively under the 35S promoter. We can see an approximate 2-fold increase in depth of imaging when comparing PFD and water. It should be noted, however, that the precise improvement seen when using PFD varies with numerical aperture of the lens and the type of tissue used. We have seen cytoplasmic streaming and root hair elongation in PFD treated seedlings, each indicative of healthy plants. In addition we have shown2 that Fv/Fm, a measure of the photosynthetic functioning of plants12 remains within tolerable limits over timescales used in imaging.
<img alt="Leaf cross-section diagram with cellular structures under microscope, includes palisade and spongy mesophyll." src="/files/ftp_upload/3394/3394fig1.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1."> 
Figure 1. Plant leaf complexity and optical implications 
Diagrammatical representation showing the anatomical features of the A. thaliana leaf in relation to the optical set-up. Abbreviations used are obj. = objective lens, imm. = immersion fluid, cov. = coverslip, mnt. = mountant, cut. = cuticle, ad. ep. = adaxial epidermis, st. = stomatal pore, sp. = spongy mesophyll, a.s. = air space, pal. = palisade mesophyll, v.b. = vascular bundle, ad. ep. = adaxial epidermis. Cell walls are indicated by black lines.
<img alt="Leaf infiltration experiment diagram showing GFP fluorescence and overlay analysis." src="/files/ftp_upload/3394/3394fig2.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2."> 
Figure 2. PFD readily penetrates Arabidopsis leaves 
(a) Leaves incubated in air, water or PFD for 5 minutes and imaged using a Leica DCF3000FX digital camera with a Leica MZ16F microscope (Leica Microsystems (UK) Ltd., Milton-Keynes, UK). The JoVE logo was printed on acetate film and illuminated from below with a light box. Exposure time was 89 ms and all images were collected and processed identically. Scale bars represent 2 mm. 
 
(b) PFD carrying purified recombinant green fluorescent protein (GFP) delimits airspaces in vivo. GFP is false coloured green, and red is used to show chlorophyll auto-fluorescence, which delimits the mesophyll cells. (Figure 2(b) reproduced with permission and full technical details available in Littlejohn et al. 20102). Scale bars represent 25 m.
<img alt="Confocal microscopy depth analysis in leaf tissue, showing fluorescence distribution per treatment." src="/files/ftp_upload/3394/3394fig3.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3."> 
Figure 3. PFD improves confocal imaging in leaves 
LSCM images showing cytoplasmically localised Venus fluorescence (green) and chlorophyll autofluorescence (red) in intact A. thaliana leaves imaged in air, water or PFD. Excitation wavelength was 514 nm and fluorescence emission was recorded at 518 nm 604 nm for Venus and at 657 nm 679 nm for chloroplasts. Each panel is a single confocal section taken from a Z-stack of 11 confocal images acquired at 5 μm intervals. These were extracted from a full Z-stack of 59 images acquired at 1 μm intervals, which have been used to produce supplementary movies. Depth measurements are given relative to the epidermal surface. Representative images, which were processed identically, are shown. Experimental details such as microscope settings are identical to those found in Littlejohn et al., 20102. Scalebars represent 20 μm.

<ol><li>Inoue, S. Foundations of Confocal Scanned Imaging in Light Microscopy. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aInoue%2C+S+%22Handbook+of+Biological+Confocal+Microscopy%2C+3rd%22">Handbook of Biological Confocal Microscopy, 3rd</a>. Pawley, J. P. , Springer Science & Business Media, LLC. New York. 1-16 (2006).</li>
<li>Littlejohn, G. R., Gouveia, J. D., Edner, C. S. mirnoff, N,, Love, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perfluorodecalin+substantially+improves+confocal+depth+resolution+in+air-filled+tissues%5BTitle%5D)+AND+%22New.+Phytologist%22%5BJournal%5D)">Perfluorodecalin substantially improves confocal depth resolution in air-filled tissues.</a> New. Phytologist. 186, 1018-1025 (2010).</li>
<li>Sargent, J. W., Seffl, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Properties+of+perfluorinated+liquids.+Fed%5BTitle%5D)+AND+%22Fed.+Proc%22%5BJournal%5D)">Properties of perfluorinated liquids. Fed.</a> Fed. Proc. 29, 1699-1703 (1970).</li>
<li>Schönherr, J., Bukovac, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Penetration+of+stomata+by+liquids%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Penetration of stomata by liquids.</a> Plant Physiol. 49, 813-819 (1972).</li>
<li>Davies, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liquid+ventilation%5BTitle%5D)+AND+%22Paediatr.+Child.+Health%22%5BJournal%5D)">Liquid ventilation.</a> Paediatr. Child. Health. 35, 434-437 (1999).</li>
<li>Shaffer, T. H., Wolfson, M. R., Greenspan, J. S., Hoffman, R. E., Davis, S. L., Clark, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Liquid+ventilation+in+premature+lambs%3A+uptake%2C+biodistribution+and+elimination+of+perfluorodecalin+liquid%5BTitle%5D)+AND+%22Reprod.+Fertil.+Dev%22%5BJournal%5D)">Liquid ventilation in premature lambs: uptake, biodistribution and elimination of perfluorodecalin liquid.</a> Reprod. Fertil. Dev. 8, 409-416 (1996).</li>
<li>Crafoord, S., Larsson, J., Hansson, L. J., Carlsson, J. O., Stenkula, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+perfluorocarbon+liquids+in+vitreoretinal+surgery%5BTitle%5D)+AND+%22Acta.+Ophthalmol.+Scand%22%5BJournal%5D)">The use of perfluorocarbon liquids in vitreoretinal surgery.</a> Acta. Ophthalmol. Scand. 73, 442-445 (1995).</li>
<li>Lowe, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+blood%3A+synthetic+substitutes+from+fluorinated+compounds%5BTitle%5D)+AND+%22Tissue+Eng%22%5BJournal%5D)">Engineering blood: synthetic substitutes from fluorinated compounds.</a> Tissue Eng. 9, 389-399 (2003).</li>
<li>Wardrop, J., Edwards, C. M., Lowe, K. C., Davey, M. R., Power, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+responses+of+plant+protoplasts+to+culture+with+oxygenated+perfluorocarbon%5BTitle%5D)+AND+%22Adv.+Exp.+Med.+Biol%22%5BJournal%5D)">Cellular responses of plant protoplasts to culture with oxygenated perfluorocarbon.</a> Adv. Exp. Med. Biol. 428, 501-505 (1997).</li>
<li>Sukumaran, N. P., Quamme, H., Weiser, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+fluorocarbons+to+study+freezing+in+plant+tissues%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Use of fluorocarbons to study freezing in plant tissues.</a> Plant Physiol. 50, 632-634 (1972).</li>
<li>Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications%5BTitle%5D)+AND+%22Nat.+Biotechnol%22%5BJournal%5D)">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li>
<li>Baker, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chlorophyll+fluorescence%3A+a+probe+of+photosynthesis+in+vivo%5BTitle%5D)+AND+%22Annu.+Rev.+Plant.+Biol%22%5BJournal%5D)">Chlorophyll fluorescence: a probe of photosynthesis in vivo.</a> Annu. Rev. Plant. Biol. 59, 89-113 (2008).</li>
</ol>

No conflicts of interest declared.

This is a simple and easy to use technique to improve microscopy of air-filled or optically complex tissues. We have shown that the technique has some strong advantages and we hope that it will be used to elucidate biological questions pertaining to air-rich tissues. For example it would be a natural choice for studies of pathogen attack in plant mesophyll or in lungs. We also are aware of the limitations of the technique. We are working on better refractive index matching, use with other modes of microscopy, and perfluorocarbon mountant replenishment during long timescale experiments. We also recognise, though, that one of the main advantages of PFCs, namely being biologically inert has a flipside. PFCs do not readily dissolve biological molecules which also implies that they cannot be easily used to deliver compounds of interest such as hormones, drugs, and other small molecules or ions.

<table><tbody><tr><td>Perfluorodecalin</td><td>F2 Chemicals</td><td>N/A</td><td>Telephone to order.</td></tr><tr><td>Carolina Observation Gel</td><td>Blades Biological</td><td>13-2700</td><td></td></tr></tbody></table>

a simple method for imaging arabidopsis leaves using perfluorodecalin as an infiltrative imaging medium

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

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.

a-simple-method-for-imaging-arabidopsis-leaves-using-perfluorodecalin

Research

JoVE Journal

Biology

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

This article provides a protocol for cultivation of Arabidopsis seedlings in the RootChip, a microfluidic imaging platform that combines automated control of growth conditions with microscopic root monitoring and FRET-based measurement of intracellular metabolite levels.

Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

Live Confocal Imaging of Developing Arabidopsis Flowers

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Here, we present a protocol for the synthesis and characterization of cerium oxide nanoparticles (nanoceria) for ROS (reactive oxygen species) scavenging in vivo, nanoceria imaging in plant tissues by confocal microscopy, and in vivo monitoring of nanoceria ROS scavenging by confocal microscopy.

Catalytic Scavenging of Plant Reactive Oxygen Species In Vivo by Anionic Cerium Oxide Nanoparticles

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

Learn to image Arabidopsis thaliana leaves using perfluorodecalin as an infiltrative mounting medium. This method doubles imaging depth compared to aqueous media while preserving living cell physiology for plant biology research.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Explore More Videos