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A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies.

Tan WK, Lim TM, Loh CS - Plant Methods (2010)

Bottom Line: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures.Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands.Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore. dbslohcs@nus.edu.sg.

ABSTRACT

Background: Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.

Results: Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®).

Conclusions: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

No MeSH data available.


Paradermal view of the salt glands of A. officinalis. Adaxial epidermal peel was obtained through an enzymatic approach. The top view of salt glands (arrows) can be viewed directly from the isolated epidermal peel using brightfield microscopy. Note the presence of chlorophyll-containing cells (dark green patches) underneath the epidermal peel that tend to obscure the salt gland images. Bar = 100 μm.
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Figure 1: Paradermal view of the salt glands of A. officinalis. Adaxial epidermal peel was obtained through an enzymatic approach. The top view of salt glands (arrows) can be viewed directly from the isolated epidermal peel using brightfield microscopy. Note the presence of chlorophyll-containing cells (dark green patches) underneath the epidermal peel that tend to obscure the salt gland images. Bar = 100 μm.

Mentions: Recent imaging trend prefers less invasive methods aiming at keeping the cells and tissues intact and alive [11,14]. The degree of light penetration through whole-mount specimens, however, limits the application of state-of-the-art microscopic techniques, such as confocal laser scanning microscopy, in obtaining deep optical sections with good cellular details from living plant tissues [11,12,14]. To solve the problem of direct visualization of salt glands from intact leaves without any clearing treatments, we enzymatically isolated adaxial epidermal peels where the salt glands are found for subsequent microscopic studies (Figure 1). Through this approach, the thickness of the leaf sections containing the salt glands was reduced, rendering paradermal microscopic observations of the salt glands possible without the need to prepare microtome leaf sections. Red autofluorescence of the chlorophyll in the mesophyll and palisade cells underneath the salt glands, however, obscured the structural observations of the salt glands on the epidermal layers when the samples were viewed under fluorescent or confocal laser scanning microscopes. Alternative approach will be required to obtain good optical sections of the salt glands using confocal laser scanning microscopy.


A simple, rapid method to isolate salt glands for three-dimensional visualization, fluorescence imaging and cytological studies.

Tan WK, Lim TM, Loh CS - Plant Methods (2010)

Paradermal view of the salt glands of A. officinalis. Adaxial epidermal peel was obtained through an enzymatic approach. The top view of salt glands (arrows) can be viewed directly from the isolated epidermal peel using brightfield microscopy. Note the presence of chlorophyll-containing cells (dark green patches) underneath the epidermal peel that tend to obscure the salt gland images. Bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2965709&req=5

Figure 1: Paradermal view of the salt glands of A. officinalis. Adaxial epidermal peel was obtained through an enzymatic approach. The top view of salt glands (arrows) can be viewed directly from the isolated epidermal peel using brightfield microscopy. Note the presence of chlorophyll-containing cells (dark green patches) underneath the epidermal peel that tend to obscure the salt gland images. Bar = 100 μm.
Mentions: Recent imaging trend prefers less invasive methods aiming at keeping the cells and tissues intact and alive [11,14]. The degree of light penetration through whole-mount specimens, however, limits the application of state-of-the-art microscopic techniques, such as confocal laser scanning microscopy, in obtaining deep optical sections with good cellular details from living plant tissues [11,12,14]. To solve the problem of direct visualization of salt glands from intact leaves without any clearing treatments, we enzymatically isolated adaxial epidermal peels where the salt glands are found for subsequent microscopic studies (Figure 1). Through this approach, the thickness of the leaf sections containing the salt glands was reduced, rendering paradermal microscopic observations of the salt glands possible without the need to prepare microtome leaf sections. Red autofluorescence of the chlorophyll in the mesophyll and palisade cells underneath the salt glands, however, obscured the structural observations of the salt glands on the epidermal layers when the samples were viewed under fluorescent or confocal laser scanning microscopes. Alternative approach will be required to obtain good optical sections of the salt glands using confocal laser scanning microscopy.

Bottom Line: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures.Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands.Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543, Singapore. dbslohcs@nus.edu.sg.

ABSTRACT

Background: Some plants inhabiting saline environment remove salts via the salt glands embedded in the epidermal tissues. Cytological studies of salt glands will provide valuable information to our understanding of the secretory process. Previous studies on salt gland histology relied mainly on two-dimensional microscopic observations of microtome sections. Optical sectioning properties of confocal laser scanning microscope offer alternative approach for obtaining three-dimensional structural information of salt glands. Difficulty in light penetration through intact leaves and interference from neighbouring leaf cells, however, impede the acquiring of good optical salt gland sections and limit its applications in salt gland imaging. Freeing the glands from adjacent leaf tissues will allow better manipulations for three-dimensional imaging through confocal laser scanning microscopy.

Results: Here, we present a simple and fast method for the isolation of individual salt glands released from the interference of neighbouring cells. About 100-200 salt glands could be isolated from just one cm2 of Avicennia officinalis leaf within hours and microscopic visualization of isolated salt glands was made possible within a day. Using these isolated glands, confocal laser scanning microscopic techniques could be applied and better resolution salt gland images could be achieved. By making use of their intrinsic fluorescent properties, optical sections of the gland cells could be acquired without the use of fluorescent probes and the corresponding three-dimensional images constructed. Useful cytological information of the salt gland cells could also be obtained through the applications of fluorescent dyes (e.g., LysoTracker® Red, FM®4-64, Texas Red®).

Conclusions: The study of salt glands directly at the glandular level are made possible with the successful isolation of these specialized structures. Preparation of materials for subsequent microscopic observations of salt glands could be achieved within a day. Potential applications of confocal fluorescence microscopic techniques could also be performed using these isolated glands. Experiments designed and targeted directly at the salt glands were explored and cytological information obtained herein could be further incorporated towards the understanding of the mechanism underlying secretion in plant salt glands.

No MeSH data available.