Document Detail

Reflection across plant cell boundaries in confocal laser scanning microscopy.
MedLine Citation:
PMID:  18778432     Owner:  NLM     Status:  MEDLINE    
The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.
D Y T Liu; B T Kuhlmey; P M C Smith; D A Day; C R Faulkner; R L Overall
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of microscopy     Volume:  231     ISSN:  1365-2818     ISO Abbreviation:  J Microsc     Publication Date:  2008 Aug 
Date Detail:
Created Date:  2008-09-09     Completed Date:  2008-10-24     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0204522     Medline TA:  J Microsc     Country:  England    
Other Details:
Languages:  eng     Pagination:  349-57     Citation Subset:  IM    
School of Biological Sciences, University of Sydney, NSW 2006, Australia.
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MeSH Terms
Cells / chemistry*
Green Fluorescent Proteins / analysis
Microscopy, Confocal / methods*
Onions / chemistry*
Plant Proteins / analysis*
Plasmodesmata / chemistry*
Recombinant Fusion Proteins / analysis
Reg. No./Substance:
0/Plant Proteins; 0/Recombinant Fusion Proteins; 147336-22-9/Green Fluorescent Proteins

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