Flower cells in 3D without SEM
Image: 3D light imaging method substitutes for problematic SEM of plant cells [Landis et al.]
US-based biologists claim to have developed a less damaging and cost-effective alternative to SEM for imaging cellular structures in plant cells.
Using a new optical sectioning method, Jacob Landis from the University of Florida and colleagues, produced high quality images of epidermal cells on the surface of flower petals with virtually no damage to the cells.
As Landis says: "This method requires no special equipment for sample preparation prior to imaging and should be seen as an alternative to SEM."
Studying flower epidermal cells is crucial to understanding the interaction between flowers and pollinators but SEM preparation and imaging compromises delicate samples.
Freeze-drying samples can render cells indistinguishable while the vacuum pressure within the SEM and the electron beam can cause additional damage.
With this in mind, Landis and colleagues developed a novel optical sectioning-3D reconstruction method that bypasses these limitations and promises a higher throughput than SEM.
As Landis explains, flower samples up to 5 cm in length were first fixed, stained and mounted on microscope slides, ready for compound fluorescence microscopy.
The researchers then used a Zeiss AxioCam high-resolution camera mounted on a Zeiss AxioPlan 2 Imaging microscope, with an Apotome optical sectioning system, to capture successive slices of the sample material.
Using AxioVision digital image processing software, the researchers acquired Z-stacks, which were then imported into Fiji, an open-source biological software platform for further cell analysis.
And crucially, the same software was used to create composite images and reconstruct 3D images with a higher resolution than SEM.
A mature flower of Saltugilia caruifolia (left), accompanied with a microscopic view of the petal lobes (right) showing the 3D cell structure. [Jacob B. Landis from: Landis, J. B., K. L. Ventura, D. E. Soltis, P. S. Soltis, and D. G. Oppenheimer. 2015]
As Landis highlights: "Cells toward the base of the petal tube were indistinguishable with SEM but clearly visible using this optical sectioning method."
The researchers believe the method can be applied to more than just petal cells, and can be used with any microscope that can produce optical sections, including confocal laser scanning microscopy and light-sheet fluorescence microscopes.
On the downside, photobleaching can take place during image capture if the intensity of the laser is too strong.
But as Landis concludes: "We believe that any kind of tissue that can be mounted on a microscope slide will be possible to image. I've spent a lot of time using SEM... and optical sectioning with 3D reconstruction is the simplest and most cost-effective method I have used."
Research is published in Applications in Plant Sciences.