The images obtained by wide-field fluorescence and LP emission filters represent the authentic fluorescence colours. For images obtained with BP emission filters, we merged the blue, green or red fluorescence channels using ImageJ Rasband — image analysis software. Details and discussions on each of the protocols for sectioning, staining and observation are provided in the following sections.
In brief, FAA typically provides adequate fixation of protein in cell walls, nuclei and other cell organelles and has been commonly used in studies by light microscopy of root and stem formation and structure. Glutaraldehyde penetrates more slowly through cell walls compared to FAA, and can be only used for small-size samples, but provides superior fixation of the ultrastructure of cell walls, cytoplasm and cell organelles.
Glutaraldehyde and OsO 4 will cross-link and further stabilize subcellular components and have been used in correlative studies by light and electron microscopy Kiernan ; Watanabe et al. These three chemicals can be applied sequentially on the same plant material, firstly FAA, then GA to provide fixation of relatively large samples, then followed by fixation with OsO 4 of subsamples for light and electron microscopy Kitin et al.
It has to be pointed out that the strong, chemically reactive fixatives are usually toxic if allowed to contact living tissue. Skin or eye contact and inhalation are the most common exposure routes. Therefore, protective wear, a fume-hood and care need to be used when working with such chemicals. Ethanol or ethanol-glycerol solutions for preservation of plant material are relatively less toxic and easier to handle.
Nakaba et al. In our experience, it is possible to increase the amount of glycerol when effects of plasmolysis do not visually alter the cell morphology and cell wall structure. Immediately after harvesting, the plant sample can be immersed in WEG in equal proportions which not only preserves the cellular structure but also the glycerol acts as a plasticizer making the suberized and lignified tissues easier to cut. The WEG preservation and storage procedure is easy to do in the lab or in the field.
We recommend to vacuum-infiltrate the sample within 2—3 h of harvesting and change to a fresh WEG because extractives from the sample may have entered the storing solution. The structures and chemical content that we have targeted for observation, such as cell wall, nuclei, starch or lipids, have been adequately preserved for light microscopy or SEM observation.
The WEG will clear the tissue from extractives, soluble pigments and non-specifically bound dye. The WEG storing solution can be easily replaced with pure glycerol as a mountant for fluorescence observations as further described in our protocols. A popular tissue-clearing agent for microscopy is chloral hydrate; however, it is a regulated drug and might be difficult to obtain.
The refractive indices of aqueous glycerol increase depending on the glycerol concentration as the following, listed by Bochert et al. All micrographs in this paper were obtained from samples that have been either stored in WEG or cryo-fixed as further described.
Transmitted-light microscopy requires thin histological sections for the light to pass through the sample. Semi-thin sections eliminate the problems of out-of-focus light that causes blur in the images; therefore, semi-thin sections can improve the imaging by both conventional light and fluorescence microscopy Kitin et al. However, these procedures require several days to be completed. Furthermore, unwanted effects by the dehydration, chemical modification or extraction of soluble substances during the steps of tissue preparation can be expected to occur.
An important advantage of the plastic-embedded sections is that fragile cellular structures, such as division plates in cambial cells, pit membranes, perforation partitions of developing vessel members or partially digested or degraded walls, remain intact in their original position. In unembedded sections prepared with a microtome or freehand, thin portions of the cell walls are often torn or displaced. Plastic-embedded sections allow for observation of the morphology and the insoluble content of vacuoles and protoplast.
However, it needs to be pointed out that many of the same advantages for preservation and observation of subtle subcellular structures are offered by cryo-sectioning, and to some extent by application of polyethylene glycol PEG embedding and preparation of clean-cut surfaces for reflected-light or epifluorescence microscopy as explained below. Microscopes with high-quality reflected-light optics or confocal microscopes are becoming more easily available in university campuses or centralized microscopy facilities.
With a wide-field fluorescence microscope, superior images can be obtained from the surfaces of samples without the need for thin sections Figs 1 through 5.
Clean and smooth surfaces can be achieved by shaving away very thin layers of the tissue with a sharp knife or a microtome blade Kitin et al. Artificial distortions of shapes or cell wall damage are less common in thick sections than in thin ones. For stabilization of soft tissues such as cambium and phloem during sectioning, some authors employ embedding in PEG as explained further in this paper.
Thick sections are suitable for 3D imaging of the morphology and cell wall development of large vascular cells Kitin et al. Making thick sections with a sliding microtome is easy to learn and requires less preparation time compared to sectioning of epoxy-embedded material. Planed surfaces are particularly adequate for plant cryo-microscopy for a detailed description of the cryo-planing procedure, see Yazaki et al.
It is considerably faster to prepare planed surfaces compared to making thin or thick sections. Visualization of lignin, suberin and extractives on microtome-planed surfaces of wood A, D, E , and on hand-cut sections B, C.
Safranin staining A and autofluorescence B through D. The red colour corresponds to more lignin. The upper arrow points to a bordered pit in the radial wall of an earlywood tracheid. The lignified walls of axial tracheids show a mixture of red and green fluorescence depending on the lignin proportion of the sectioned wall layer, middle lamella red or S2 green.
The middle arrow points to ray tracheids, and the lower arrow points to a lignified ray parenchyma cell indicated with red colour and thick wall. B Hand-cut, transverse section of a young poplar stem Populus sp. Green is emitted from lignified walls and red from chloroplasts in ray parenchyma.
The orange-yellow colour is a mixture of red and green indicating that the horizontal wall of ray cells is near the surface of the section. C Transverse cut through the cortex of a 1-year-old poplar stem Populus sp. The uneven focus is due to the rough surface of the sectioned cortex that contains cell types with contrasting density and hardness see the text for discussion on hand sections. The arrow at the left points to blue fluorescence from cork. The two arrows at the right point to blue fluorescence from lignified sclerenchymatic cells.
The red fluorescence is emitted from chloroplasts in cortical parenchyma and rays. A small portion of last-formed xylem weak, blue fluorescence can be seen at the right side of the micrograph.
D Transverse-cut surface of wood of a low-lignin hybrid poplar tree Kitin et al. The sample was cryo-fixed and cut in the frozen state, then visualized in the frozen state by wide-field fluorescence without staining blue excitation and band-pass emission BP —; Nikon; Eclipse E Xylem areas that are not functional for water conduction nxy contain xylem vessels filled with phenolics arrows while no occlusions are visible in the conductive xylem cxy. Arrows point to xylem vessels occluded with phenolics.
After freeze-drying, the phenolics emit strong blue and green fluorescence. A single-track, two-channel image with and nm laser lines and band-pass filters BP —, BP — The image is a maximum projection of four optical sections at 1-mm intervals. An interesting technique of polishing the surfaces of epoxy-embedded tissue blocks followed by CLSM is described by Dickson et al. Sanded wood surfaces can be also appropriate for reflected-light microscopy as described by Cerre and Hunt et al.
Tissue blocks or thick sections allow for large-area observations which can be especially useful in studies of roots and stems, and in particular, when we need to scale up from the subcellular to the tissue- or organ-level investigation. For reflected-light or epifluorescence microscopy, the quality of observation depends mainly on the smoothness of the surface of the section and less on its thickness.
Light colour is indication of increased surface roughness and light scattering, while surfaces become darker and reflective as they become smoother. For example, a light-coloured sawn surface becomes much darker as damaged cells are polished or cut away. Microtomes are designed to provide sections with precisely determined and uniform thickness. Vibratomes are helpful for cutting soft plant organs such as leaves, shoots and young roots Gunawardena et al. Sliding microtomes are great tools for cutting series of sequential sections for 3D reconstructions of plant vasculature Zimmerman and Tomlinson ; Kitin et al.
Sliding microtomes have also been successfully employed for planing of wood surfaces for ecological or functional studies of xylem structure Utsumi et al. Sliding microtomes are suitable for cutting samples with rigid cell walls and relatively uniform hardness such as lignified or suberized tissues. However, making even cuts through tissues that contain high proportions of unlignified cells is problematic, and therefore, embedding of the tissue in media that can stabilize the soft cells is often implemented.
Embedding in paraffin or epoxy is associated with the use of rotary microtomes and preparation of ultra-thin or semi-thin sections. For preparation of thick sections with a sliding microtome, embedding in PEG has been successfully employed as explained further in this paper.
Freehand sectioning is faster, cheaper, easy to learn and can provide high-quality sections for light microscopy. Freehand sections have been routinely used in many laboratories in particular for studying roots and leaves or xylem and phloem.
A variety of techniques can be employed for cutting plant material by freehand Ruzin ; Wiedenhoeft ; Yeung et al. We use a cutting board and a sharp razor blade or scalpel to cut thin slices of roots or stems. Cutting while observing the sample under a dissecting microscope is helpful for controlling the precise position and direction of the cut. Cutting gently with slightly advancing movement of the blade may help achieve fairly thin and flat sections with intact arrangement of cortical and vascular tissues.
Cutting small areas reduces the overall cutting force and typically results in better sections. Furthermore, young and unlignified roots or stems can be cut after PEG embedding, as described below, which stabilizes the sample from either outside and inside as low molecular weight PEG easily infiltrates plant tissues.
For fully lignified tissue, each section of razor blade can often only make one premium quality cut. An important note to be made is that before freehand or microtome sectioning, FAA or other fixative media has to be well washed out from the specimens. We recommend rinsing FAA-fixed samples for 15—30 min in running tap water before cutting sections for microscopy.
The plant material has to be wetted with water or glycerol before sectioning except for PEG-embedded material see notes in the following paragraph. Depending on the hardness of the tissue, blades may have to be frequently changed. Biological sample embedment in PEG carbowax for light and electron microscopy has long history in both plant and animal anatomy.
Polyethylene glycol is soluble in water and can be easily removed from the sections before staining and observation. For this reason, PEG embedding has been useful for immunostaining or in situ hybridization of sections, unlike the difficult to remove impregnation with waxes or resins Clayton and Alvarez-Buylla Ferreira et al.
However, for special applications such as immunostaining, there have been concerns about using higher molecular weight PEG because high temperatures may compromise immunoreactivity. Gao and Hayat provide discussions of different PEG embedment techniques in various studies. More recently, PEG embedment was applied in studies of xylem and bark development in plant stems Schmitz et al.
Before sectioning, Barbosa et al. As we discuss later, PEG solution has been used as a carrier of the fluorescent stain FY for labelling of root endodermis Brundrett et al.
They suggested fixation with ferrous sulfate and formalin previous to PEG embedding for detection of total phenolics Ferreira et al. We use the following procedure for embedding root and stem specimens in PEG The chemically fixed specimens are rinsed for 15—30 min in running tap water. The time of infiltration with liquid PEG depends on the size and nature of specimens; for example, larger than 1 cm 3 specimens with suberized tissue may require longer time for successful infiltration, such as 2—3 days.
Finally, the preparations can be transferred to embedding moulds at room temperature for curing the PEG and the sample blocks will be ready for sectioning.
Smearing a thin layer of Vaseline on the surface of the moulds beforehand can help to easily separate the blocks from the moulds. Store the embedded material in air-tight containers to prevent rehydration. Current methods of cryo-microscopy are reviewed in depth elsewhere Echlin ; McDonald and Auer ; Kitin et al.
Cryo-fixation preserves the original cellular structure, water content and position of secondary metabolites or tracer dyes within cell lumens. Frozen specimens can be cut or planed with a cryo-microtome and then observed in the frozen state by cryo-electron microscopy Utsumi et al. Cryotomes for sectioning of frozen samples are particularly useful in studies of soft tissue and these instruments have been often employed in animal research.
Cryo-sectioning preserves the subtle cell walls and subcellular structures similarly to the method of cutting plastic-embedded material. Furthermore, rapid-freezing followed by cryo-sectioning preserves the cell water content and distribution of secondary metabolites. Figure 1D shows phenolic deposits in xylem vessels which would have been extracted from the section by conventional sample preparation. Extractives typically cannot be studied in conventional thin sections because they are lost during sectioning and washing of the sample Kitin et al.
Cryo-fixation of extractives as well as sections free from extractives can both provide important information. For example, the location of soluble metabolites is visible in Fig. The cryo-fixed samples can be freeze-dried to avoid the inconvenience of maintaining frozen samples. A freeze-dried sample observed with a short-distance objective lens and 3D optical sectioning is shown in Fig.
Visualization of lignified and non-lignified cellulosic cell walls in thick sections stained with calcofluor A or with safranin-calcolfluor B, C. A Cellulosic walls blue-green fluorescence of gelatinous fibres gf , ray cells and tyloses in wood of a low lignin poplar mutant. Red autofluorescence is emitted from the lignified vessel wall showing bordered pits. Tyloses vertical arrows ; ray cells horizontal arrows on the left ; xylem vessel wall with bordered pits large arrow ; and membranes of parenchyma-to-vessel pits horizontal arrows on the right are shown.
B Transverse section of poplar wood Populus tremuloides visualized by wide-field fluorescence Olympus BX60; — excitation, LP emission. Red fluorescence from lignified walls is visualized simultaneously with blue fluorescence from cellulosic portions of the walls. The arrows point to non-lignified pit membranes of half-bordered pits between ray parenchyma and xylem vessels. Weaker blue signal is emitted from non-pit portions of the lignified walls of fibres where cellulose was exposed to calcofluor staining.
The cellulose in lignified walls can become accessible to staining in damaged wall areas such as small cracks that are caused during sectioning. The section was inoculated with the white rot fungus Phanerochaete chrysosporium which removes lignin from cell walls.
The arrow points to fluorescence from calcofluor-stained cellulose in a delignified xylem ray 20 days after inoculation. This image was acquired by merging the fluorescence signal from calcofluor-stained cellulose — excitation, LP emission with the red fluorescence signal from safranin-stained lignified walls green excitation, LP emission.
Green colour was artificially assigned to the calcofluor signal for a higher contrast for more details, see the text for discussion on multichannel imaging. The combination of green and red provides a clearer view of the cellulose exposure that occurs predominantly in the rays and in the radial walls pit regions of earlywood tracheids.
More details on different methods of plant cryo-microscopy are provided by Yazaki et al. Epifluorescence microscopy enables convenient observation of sectioned or planed surfaces of relatively large tissue samples. Furthermore, by fluorescence CLSM, large plant cells, such as xylem vessel elements, or large areas of the tissue can be observed in 3D on both the tissue level and subcellular detail Kitin et al.
Some fluorescence microscopes can accommodate cryo-stages that allow for observation of samples in the frozen state Kitin et al. Note that fluorescence spectra can be influenced by molecular environments, such as temperature, pH or concentration and interactions between fluorophores or other chemicals present.
Therefore, quantitative fluorescence microscopy is challenging, especially for comparisons across different dates, sites or using different microscopy equipment. Techniques to improve the accuracy and precision of quantitative fluorescence are available Pawley ; Waters Chlorophyll, cutin, suberin and various polyphenols including lignin are naturally fluorescent substances in plant cells Rost ; Hutzler et al.
Autofluorescence indigenous fluorescence can be a significant problem when it overlaps with the fluorescence label on structures targeted for observation. A number of techniques are available to suppress the unwanted fluorescence signal. Application of non-fluorescent stain such as bromphenyl blue for quenching the autofluorescence is one example. Inspection of the fluorescence excitation and emission spectra of the different parts of the sample can determine whether unmixing is likely to succeed.
Another way is to extract the autofluorescing substances by using clearing agents. Dalton et al. On the other hand, autofluorescence can be very useful in plant anatomical studies as it allows fluorescence imaging without staining Fig. For example, different compositions of lignin have different natural fluorescence characteristics and the strength of the fluorescence signal increases with lignin concentration Albinsson et al.
Donaldson showed that the autofluorescence emission profiles of fibre wall and xylem vessel wall in poplar are different. In addition to the typical role of matching index of refraction, mounting media for fluorescence studies should not obscure the fluorescence signal. Donaldson recommended glycerol at pH 9 as the optimal mounting medium for lignin spectroscopy at the visible excitation range. According to the same author, thiodiethanol, which has a higher refractive index than glycerol, allowed a stronger fluorescence signal as a mounting medium with UV excitation.
Strong fluorescence from cytoplasm and primary cell walls can be induced by some fixatives such as glutaraldehyde or WEG. Glutaraldehyde-induced fluorescence was used by Singh et al. The knowledge of autofluorescence from various plant substances can have important applications in plant histochemical and cell developmental studies; therefore, more investigations of the nature of autofluorescence are desirable.
Understanding autofluorescence can be particularly useful for direct observation of frozen plant material after cryo-sectioning. Advanced techniques such as fluorescence resonance energy transfer FRET and fluorescence lifetime imaging FLIM have been applied in studies of the interactions of enzymes or fluorophores with complex lignocellulosic walls Donaldson and Radotic ; Houtman et al. Lignin exhibits autofluorescence over a large spectral range, which has been used for imaging the structure of wood and in studies of delignification or degradation of woody cells Donaldson et al.
Here, we illustrate an easy way for differentiation between lignified and unlignified cellulosic walls by using the natural fluorescence of lignin in combination with staining of polysaccharides. Cellulose is the main building material of plant cell walls and even strongly lignified walls may contain more cellulose than lignin. The mechanisms of cellulose staining with different categories of dyes are reviewed by Wallace and Anderson and Hubbe et al. Recently, the fluorophores CR and pontamine fast scarlet 4B PFS synonym, direct red 23 have gained popularity because of their affinity to cellulose and chitin Slifkin and Cumbie ; Verbelen and Kerstens ; Hoch et al.
It is important to keep in mind that most dyes bind by association with their target. Even highly specific protein-binding domains have cross-reactivity. The association of dyes with their targets is usually less specific and so will have even more cross-reactivity. A dye found to be selective in one environment will be non-selective in the presence of a different mounting medium or other chemical binding sites in a different sample.
For example, the membrane dye FM Thermo Fisher Cat T clearly shows fungal membranes and not bleached kraft paper fibres. However, when fibres contain large quantities of oxidized lignin, FM is selective for the fibre Fig. Another common example is the claim of specific dye affinity to chitin or cellulose. In almost all these cases, the specificity is observed because the samples do not contain both targets. In our experience, CR molecular weight is convenient to use because staining is stable in water and glycerol while unbound dye is quickly removed from the sections.
The fluorescence of diluted CR in water and alcohol is very weak and practically the microscopic preparations are free from unwanted background fluorescence. The absence of fluorescence contamination is an important advantage particularly for imaging thick sections that are difficult to clear after staining.
In the s, CR was commonly used to stain cotton but was later abandoned due to toxicity. It is believed that adsorption binds CR to cellulose when its molecules align along the linear molecules of crystalline cellulose Woodcock et al. CR, similar to calcofluor, reportedly has affinity to a larger class of polysaccharides, including glucans, and xyloglucans Wood et al. Congo red stains lipopolysaccharides of Gram-negative bacteria and also has become the classic histochemical probe for amyloids in Alzheimer disease research Steensma ; Yakupova et al.
Variations in intensity of CR staining should be considered with caution because of the bifluorescence of this dye, i. The bifluorescent property of CR has been used for studying the microfibrillar organization of secondary cell wall using polarized optics Verbelen and Kerstens Another property of the CR dye that needs to be considered is that under white light illumination its colour changes from blue to red with the increase of the pH from 3.
This metachromatic property may offer applications for detection of acidity or for multi-staining. Congo red is possibly carcinogenic and mutagenic and protective wear should be used when working with this dye. We use the following protocol of staining with CR and fluorescence microscopy. FAA-fixed sections are firstly washed in running tap water for 30 min.
Then, a 0. The sections are rinsed two times in distilled water, then placed on a glass slide in a drop of water or aqueous glycerol for observation by fluorescence microscopy. Sections can stay for long time in glycerol without any visible leaking of CR in the background. Lignified, suberized and cellulosic walls in hand-cut transverse sections of root of a black mangrove plant Bruguiera gymnorrhiza visualized by wide-field fluorescence Olympus BX A Fluorol yellow staining for visualization of suberized endodermis arrows.
Inner cortex ctx , and xylem xy are visualized with the blue autofluorescence of lignified walls — nm excitation, LP emission. Note the stronger red fluorescence from phloem vertical arrow. Xylem, phloem sclerenchyma and lignified thickenings of cortical parenchyma cells are visualized with the blue autofluorescence of lignin — nm excitation, LP emission.
Merged green and red channels BP —; BP — D Acridine orange staining showing lignified walls green fluorescence in xylem and cortex. A combination of transmitted white light and wide-field fluorescence — nm excitation, LP emission was used to show the parenchyma cells and the intercellular air spaces in the cortex.
E The same section as in D but visualized only by fluorescence without the transmitted light. Note the blue autofluorescence of Casparian thickenings arrow in the endodermal cells that at an early stage of development are not stained with the acridine orange. The images in Fig. The green channel reveals the autofluorescence of lignified cell walls, which combined with the red signal of CR produces a strong contrast between lignified and primarily cellulosic walls Fig.
Developing xylem cells prior to lignification are stained more strongly with CR than the parenchyma of cortex and pith Fig. Probably, the stronger staining with CR is facilitated by the occurrence of various polysaccharides and proteins in the cell walls and cytoplasm of differentiating xylem.
Cellulose or other polysaccharide components are also present in the lignified walls; therefore, CR will stain to some extent the lignified walls too Hubbe et al. Consequently, a single red-channel observation may not be useful for differentiation of lignified and non-lignified walls. It can be seen in Figs 4 and 5 that the contrast between unlignified and lignified walls is effectively enhanced by using the combination of green and red colours see also Fig.
A greater specificity can be achieved by immunological staining where highly specific interactions between fluorescently labelled antibodies and antigens are used. Immuno-labelling has been applied in studies of complex lignocellulosic materials by many authors Funada et al.
Novy et al. As hundreds of carbohydrate-binding domains, many already fluorescently labelled, are commercially available, this represents an excellent toolbox for investigating carbohydrate localization, as long as the carbohydrates are accessible to the rather large proteins. Hand-cut sections of root and stem of a black mangrove plant B. A Longitudinal-oblique section through the root endodermis arrows showing xylem inside the endodermal cylinder. Staining with fluorol yellow UV excitation and LP emission.
Suberized cell walls are yellow or mixed blue-yellow and lignified walls xylem and cortex emit blue-green autofluorescence.
B Transverse section of a root showing the cortex, epidermis upper arrow and endodermis lower arrow. The composite image showing red staining of cellulosic walls and green autofluorescence of lignin and suberin. C Transverse section of a root showing inner cortex, endodermis, phloem and xylem. Note the green autofluorescence of lignified thickenings of cortical parenchyma cells left arrow.
The right arrow points to a transfusion endodermal cell with no suberin and lignin in the cell wall. The same staining and imaging conditions as in B. D Transverse section through the hypocotyl of the same plant as in C showing inner cortex, xylem and small portion of the pith. Developing xylem cells with unlignified walls vertical arrow are stained stronger in bright red. The small arrow points to lignified cells on the outer side of the phloem.
The green and red emission from fluorol yellow-stained suberized walls is considerably stronger than the green and red from lignified and non-lignified walls, respectively. The resulting image shows red staining of polysaccharides, yellow overlapped green and red for suberized cell walls and green for lignin. A A lateral rootlet obliquely cut shown at the outer region of the cortex of the mother root. The large arrow points to yellow-stained endodermis of the rootlet.
Note the blue autofluorescence of xylem inside the endodermal cylinder of the rootlet. The blue and yellow fluorescence of exodermal tissue cork indicates presence of lignin and suberin. Blue fluorescence is also emitted from lignified astrosclereids in the cortex small arrow. Non-lignified cell walls are stained in red.
The arrow points to a transfusion endodermal cell with no suberin or lignin in the cell wall. The autofluorescence of lignified walls of xylem and sclerenchymatic cells in the cortex is shown in green. The arrows on the left point to exodermis.
The vertical arrow points to the endodermis of the rootlet. Note the autofluorescence of xylem inside the endodermal cylinder green. Phloem and parenchymatic cells with unlignified walls Congo red staining occupy the space between the xylem and endodermis. Note also the loose arrangement of cellulosic parenchyma cells in the cortex of the mother root resulting in formation of intercellular air spaces.
Calcofluor white M2R molecular weight has been used as a fluorescent brightener in the paper and textile industry, as well as for studying cell walls of fungi, bacteria and plants Hughes and McCully ; Galbraith ; Wood et al.
When excited with UV light or laser, calcofluor white produces blue fluorescence of cellulosic walls Donaldson and Bond ; Kitin et al. The protocol by Nakaba et al. Figure 2A shows the unlignified walls of gelatinous fibres, ray cells and tyloses in blue, in contrast to the red autofluorescence of the lignified wall of a vessel element. Furthermore, the confocal image in Fig.
The half-bordered contact pits in Fig. The formation of parenchyma-to-vessel contact pits in poplar was studied by Murakami et al. We use the following protocol of staining and fluorescence microscopy.
The calcofluor staining procedure should start with washing fixed sections in running tap water for 15—30 min. Then, stain with a 0. Rinse the sections with distilled water after the staining. Thick sections should be rinsed at least 10 times for removing the excess calcofluor. Temporary microscope slides can be prepared by mounting in water or glycerol.
Note that calcofluor is often sold in combination with a small amount of blue dye often Evans blue to quench unwanted background fluorescence. Safranin molecular weight is an inexpensive, general stain for histological sections and is available in most research laboratories. It has a long history of applications and continues to be popular today for studies of plant cellular structure and for visualizing xylem cells Kitin et al.
Sections can be quickly stained with 0. The time-consuming procedure with gradual ethanol series is aimed to preserve the morphology of subcellular structures and has been adapted from transmission electron microscopy TEM protocols. It allows differentiating cell walls or protoplasmic content to be studied by correlative light and electron microscopy. Moreover, the continuous rinsing in ethanol solutions will clear thick sections not only from the excess dye but also from some pigments and extractives which will improve the clarity of cell wall observations.
Then follow the calcofluor staining procedure as described earlier. Sections stained with safranin can be immersed in water or immersion oil for preparation of temporary microscope slides Donaldson and Bond ; Bond et al. Sections can be also observed in aqueous glycerol; however, they must be rinsed in fresh aqueous glycerol immediately before observation because safranin tends to leach out and produce fluorescent background in glycerol mounting medium.
The results are blue fluorescence from cellulosic portions of the walls and green or red fluorescence from lignified walls. The calcofluor signal in Fig. In Fig. The exposed cellulose in the xylem cell walls in Fig. The calcofluor signal is stronger in the earlywood ew which indicates that the white rot fungus degrades lignin faster in the ew than in the latewood lw. Depending on the concentration of binding sites, interaction between dye molecules may lead to shifts in the intensity or spectrum of fluorescence due to quenching or FRET.
Therefore, various degrees of lignification in different cell types and cell wall layers can be qualitatively visualized with safranin staining Fig. Similarly, the autofluorescence of conifer xylem cells is stronger in the middle lamella and cell corners where the lignin proportion is higher Donaldson Besides safranin, other green or red fluorescent dyes that can produce contrast to calcofluor-stained cellulosic walls include AO, acriflavine, basic fuchsin and crystal violet Donaldson et al.
By contrast, acidic negatively charged dyes, such as acid fuchsin, tend not to stick to lignified cell walls, in our experience. The electrochemical binding between the cationic dye AO and cell wall has been studied stoichiometrically for analysis of the chemical changes during fungal biodegradation of wood Houtman et al. Acridine orange emits green as a single molecule and red as a dimer.
Therefore, when the metachromatic properties are desired, such as staining acid groups on biomass, it has been applied at a concentration where both monomers and dimers were stable Houtman et al. Others have achieved the same result by putting AO on at high concentration, then extensively rinsing in aqueous ethanol so only the most stable dimers remained Nakaba et al. The micrographs in Fig. Similarly to safranin, AO may stain non-lignified walls either by intercalation green fluorescence or by bonding with negatively charged carboxylic functional groups red fluorescent dimers Houtman et al.
Nevertheless, clear differentiation between lignified and cellulosic portions of cell walls is achieved because of the considerably weaker fluorescence of safranin-, or AO-stained polysaccharides, relative to the blue calcofluor signal Fig.
For more discussion on the mechanisms of staining involving two dyes for colour differentiation of lignin domains in cellulosic walls, see Hubbe et al. Combining different imaging techniques may reveal important details. For example, in Fig. The parenchyma of the cortex and intercellular spaces can be also seen in combined fluorescence and transmitted-light images Fig. However, the Casparian thickenings of the endodermis are not visible in Fig.
The fact that the Casparian thickenings are not stained with AO suggests that the lignocellulosic complex in the endodermal cell wall is different from those in the xylem and cortical cells, at least in this stage of root development Fig. A large number of fluorescent probes for lipids and cell membranes have been developed mainly for medical research as lipophilicity is an important property of drugs.
Berberine hemisulfate BH and FY are commonly used fluorescent stains for lipids and suberin in the traditional plant anatomy Brundrett et al. Make your longitudinal sections here. Tracheary Elements in Secondary Xylem The basic difference between tracheids and vessel members is the presence of a perforation plate on the end walls of vessel members and its absence on tracheids. The perforation plate has openings that are larger than the pits that are present in tracheids.
A linear series of vessel members is called a vessel. The secondary xylem wood is highly complex and will be studied in greater detail in later labs. For now it is sufficient to be introduced to the basic difference between Tracheids and Vessel Members. Make or observe free-hand cross sections of Podocarpus which is a Gymnosperm and Coffee Coffea which is an Angiosperm. Examine unstained with polarizers. Locate the xylem that should be highly birefringent.
Stain with phloroglucinol. The xylem is a large continuous zone and it should stain red-orange. The phloem of Coffea has fiber bands that are birefringent , but are discontinuous. How can you locate the phloem in Araucaria? Compare the xylem in Podocarpus and Coffea. Can you see any differences in the size of cell diameters within each?
In other words, which is more homogenous in cross section? The torus is more darkly stained and fairly easy to spot. View macerated Pine wood and note the relative uniformity of the cells which are all Tracheids.
Perforation plates are NOT present on the end-walls of the Tracheids. However, large pits may be clustered where tracheids overlap. Radial section of Pinewood viewed with crossed polarizers. Unlike tracheids, the vessel members have large openings in their end walls; in this case, Simple Perforation Plates one opening per end wall.
Find these openings. Use longitudinal sections to see the numerous bordered pits on the sidewalls of the vessels. These have simple perforation plates. Which type would be most efficient for water conduction? Look also for Vessel Members and whole individual vessels in macerated Oak wood. The detailed structure of sieve elements in the phloem cannot be observed easily without the use of special staining techniques.
Consequently, some of the material used in this exercise will be fresh. Sections of living material are usually more difficult to interpret than commercial slides. Primary Phloem of squash Cucurbita Study prepared slides of cross and longitudinal sections of Cucurbita stems.
Locate the xylem and phloem. Indeed, xylem function decays from relatively early in the season, which mirrors the decline in calcium import Wilkinson, ; Jones et al. This naturally occurring mechanism could be principally responsible for the increased phloem inflow to the fruit that maintains growth Lang, It has been shown in apple that the direction of xylem movement reverses diurnally such that sap flow is from fruit to tree during the day and from tree to fruit at night Lang, ; Lang and Volz, Programmed xylem breakdown is likely to reduce diurnal apoplastic backflow of solutes from fruit to the tree , which promotes the partitioning of assimilates to reproductive sinks, as suggested for grapes Lang et al.
A rather variable timing of xylem dysfunction could, therefore, create high variability in fruit mineral composition. Furthermore, the reduction in dye accumulation towards the calyx end of the fruit fits with the longitudinal gradient in calcium concentration found in apple fruit Ferguson and Watkins, Thus the localized nature and the susceptibility to the disorder fit with the spatial and temporal profile of dye accumulation in the fruit and, by implication, with xylem functionality.
Fruit were assessed at 64 and 67 DAFB, respectively. The degree of coloration in fine vascular anastomoses indicates a difference in vascular function between the two fruit. The mean dye level for each time period was obtained by assessing 15 fruit per cultivar. Vertical bars represent s. A Breakage of the xylem strand. Shattered parts are displaced so that parenchyma cells occupy the resulting void. B Ruptured string of vessels.
Multiple breaks signify excessive change in length of the stressed walls. Arrows indicate the structural changes being described. Aloni R. Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures. Planta : — Aloni R , Barnett JR. The development of phloem anastomoses between vascular bundles and their role in xylem regeneration after wounding in Cucurbita and Dahlia.
Developmental anatomy of vascular tissues in York Imperial apple, with special emphasis on the pedicel. Evidence for xylem discontinuity in Pinot noir and Merlot grapes: dye uptake and mineral composition during berry maturation.
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