Related Experiment Video
Updated: Jul 1, 2026
09:20
Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells
Published on: August 11, 2020
Reflection across plant cell boundaries in confocal laser scanning microscopy
D Y T Liu1, B T Kuhlmey, P M C Smith
1School of Biological Sciences, University of Sydney, NSW 2006, Australia.
Journal of Microscopy
|September 10, 2008
Summary
Confocal microscopy of green fluorescent protein (GFP)-tagged proteins in plant cells can create artifactual "doublets" across cell walls. These optical artifacts, not intercellular movement, explain paired fluorescent spots, challenging plasmodesmata localization studies.
Area of Science:
- Plant Cell Biology
- Microscopy Techniques
- Molecular Imaging
Background:
- Green fluorescent protein (GFP) tagging and confocal laser scanning microscopy are standard for determining plant cell sub-cellular localization.
- Plasmodesmata are often identified by punctate labeling across plant cell walls, with paired puncta ('doublets') suggesting specific localization.
- Biolistic bombardment typically transforms single plant cells, making observed doublets across cell boundaries unexpected without intercellular protein movement.
Purpose of the Study:
- To investigate the nature of paired fluorescent spots ('doublets') observed at cell boundaries in biolistically transformed plant cells.
- To determine if observed doublets indicate intercellular movement of GFP-tagged proteins or are an artifact of imaging.
- To assess the influence of the plant cell wall on the optical properties of confocal microscopy.
Main Methods:
- Expression of a green fluorescent protein (GFP)-tagged putative plasmodesmal protein in Allium porrum epidermal cells via biolistic bombardment.
- Confocal laser scanning microscopy to observe fluorescence patterns at cell boundaries.
- Finite-difference time-domain (FDTD) optical simulations to model light behavior near the plant cell wall.
Main Results:
- Observed doublets were formed by single fluorescent spots near the cell boundary, visible only at specific focal planes.
- Fluorescence from the portion of the doublet outside the transformed cell exhibited polarization.
- Optical simulations revealed significant distortion of the confocal microscope's point spread function due to refractive index mismatches at the cell wall, causing out-of-focus light detection.
Conclusions:
- The appearance of doublets across plant cell boundaries is an optical artifact caused by the plant cell wall's effect on light.
- Artifactual doublets arise from the distortion of the confocal microscope's point spread function and the scattering/reflection of light by the cell wall.
- Observed doublets are insufficient evidence for plasmodesmal localization of GFP-tagged proteins, necessitating re-evaluation of previous findings.
Related Concept Videos
01:16Confocal Fluorescence Microscopy
Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
01:05Total Internal Reflection Fluorescence Microscopy
Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
01:37Super-resolution Fluorescence Microscopy
Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been developed.
01:28Three-Dimensional Microscopy in Microbiology
Three-dimensional imaging techniques are essential in cell biology, allowing researchers to visualize intricate cellular structures with high resolution. Two prominent methods, Differential Interference Contrast Microscopy (DIC) and Confocal Scanning Laser Microscopy (CSLM), provide distinct advantages for imaging live and thick specimens, respectively.Differential Interference Contrast MicroscopyDIC microscopy enhances contrast in transparent, unstained samples by converting phase...