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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.


Related in: MedlinePlus

Confocal microscopic images of isolated A. officinalis salt glands showing the degree of red autofluorescence. (A) Image of isolated salt glands showing red autofluorescence (faintly visible). (B) Image of isolated salt glands captured simultaneously in the transmission and fluorescent modes. Excitation wavelength was 543 nm (100%) and signals in the red wavelength range were captured with Zeiss LSM 510 using high pass filter 560 nm. Bars = 20 μm.
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Figure 8: Confocal microscopic images of isolated A. officinalis salt glands showing the degree of red autofluorescence. (A) Image of isolated salt glands showing red autofluorescence (faintly visible). (B) Image of isolated salt glands captured simultaneously in the transmission and fluorescent modes. Excitation wavelength was 543 nm (100%) and signals in the red wavelength range were captured with Zeiss LSM 510 using high pass filter 560 nm. Bars = 20 μm.

Mentions: Isolated salt glands excited with a 543 nm laser showed only faintly visible red autofluorescence when the excitation intensity was increased up to a maximum of 100% (Figure 8). Naturally occurring fluorochromes like chlorophyll were reported to produce red autofluorescence when excited by a 514 nm Ar ion laser [18,20,21], which also explains the high background fluorescence observed from the isolated epidermal peels with chlorophyll-containing mesophyll-palisade cell layers underneath. The absence of red autofluorescence in A. officinalis salt gland suggests that compounds like chlorophyll might be absent in these cells. Previous reports also revealed the absence of functional chloroplasts in all species of salt glands except for the bladder cells of Atriplex [1,9]. Further salt gland studies that included the use of fluorochromes with emission spectral in the red wavelength range were thus possible.


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)

Confocal microscopic images of isolated A. officinalis salt glands showing the degree of red autofluorescence. (A) Image of isolated salt glands showing red autofluorescence (faintly visible). (B) Image of isolated salt glands captured simultaneously in the transmission and fluorescent modes. Excitation wavelength was 543 nm (100%) and signals in the red wavelength range were captured with Zeiss LSM 510 using high pass filter 560 nm. Bars = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Confocal microscopic images of isolated A. officinalis salt glands showing the degree of red autofluorescence. (A) Image of isolated salt glands showing red autofluorescence (faintly visible). (B) Image of isolated salt glands captured simultaneously in the transmission and fluorescent modes. Excitation wavelength was 543 nm (100%) and signals in the red wavelength range were captured with Zeiss LSM 510 using high pass filter 560 nm. Bars = 20 μm.
Mentions: Isolated salt glands excited with a 543 nm laser showed only faintly visible red autofluorescence when the excitation intensity was increased up to a maximum of 100% (Figure 8). Naturally occurring fluorochromes like chlorophyll were reported to produce red autofluorescence when excited by a 514 nm Ar ion laser [18,20,21], which also explains the high background fluorescence observed from the isolated epidermal peels with chlorophyll-containing mesophyll-palisade cell layers underneath. The absence of red autofluorescence in A. officinalis salt gland suggests that compounds like chlorophyll might be absent in these cells. Previous reports also revealed the absence of functional chloroplasts in all species of salt glands except for the bladder cells of Atriplex [1,9]. Further salt gland studies that included the use of fluorochromes with emission spectral in the red wavelength range were thus possible.

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.


Related in: MedlinePlus