The authors would like to thank the Maize Genetics Cooperation, the Maize Cell Genomics Project, Dave Jackson (Cold Spring Harbor Laboratory, NY), Anne W. Sylvester (Marine Biological Laboratory, University of Chicago, IL), Andrea Gallavotti (Rutgers University, NJ), and Carolyn G. Rasmussen (University of California, Riverside) for providing the mutant and transgenic stocks, as well as Robert F. Baker and Alexander Jurkevich of the Advanced Light Microscopy Core at the University of Missouri-Columbia for their assistance with confocal microscopy. JMR was supported by the J. William Fulbright Fellowship, The Diane P. and Robert E. Sharp Fund, and the National Science Foundation&#39;s Plant Genome Research Program (IOS-1546873) to PM.&#160;CDTC, CMRV, EDCDP, and RJRR are supported by the Ateneo College Scholarship Program. CDTC, EDCDP, and RJRR are supported by the DOST-SEI S&#38;T Undergraduate Scholarship. DODL is supported by Fr. Thomas Steinbugler SJ Academic Scholarship. RJRR is supported by Aiducation International&#8211;Pathways to Higher Education Scholarship. This work was supported by the School of Science and Engineering and the Rizal Library, Ateneo de Manila University.

Improved Methods for Imaging Maize Leaf Primordia

The authors do not have any conflicts of interest to disclose.

We present two methods for preparing maize leaf primordia for cellular imaging. The first method (protocol section 2) allows measurement of the primordium for transverse section analysis, while the second method (protocol section 3) enables unrolling and flattening of the primordium for whole-mount analysis. These methods facilitate the cellular imaging of FPs in maize leaf primordia24 (as shown in Figure 4 and Figure 5) and provide simple solutions to the challenges of imaging developing maize leaves. Protocol section 2 reduces dissection time and improves sampling accuracy by measuring the primordia prior to sectioning rather than relying solely on staging parameters9,16. With commercially available nano tape, protocol section 3 solves the long-standing problem of imaging whole-leaf primordia in maize. This protocol improves on the previous method, which used dialysis tubing31, and is a much cheaper alternative to CT and MRI11,32,33. However, when it comes to visualizing leaf anatomical traits and producing optimal results, both protocols have some limitations, which are outlined in Table 2 and are discussed in more detail below.
In protocol section 2, we encountered difficulty visualizing cell outlines in thick transverse sections of the leaf primordia, and counterstaining with cell wall- or plasma membrane-binding fluorescent dyes did not provide satisfactory results. For instance, FM 4-64 produced suboptimal results compared to the plasma membrane FP marker, pZmPIP2-1::ZmPIP2-1:CFP39 (PIP2-1-CFP; Figure 3A-D). To overcome this limitation, we recommend using a vibratome to produce thinner tissue sections (~0.1 mm)58 which will allow for a vivid bright-field imaging of cell outlines or optimizing the counterstaining protocol47,59.
In protocol section 3, the main limitation is the difficulty of mounting the leaf without tearing, damage, or air bubbles, as detailed in protocol steps 3.2.5-3.2.6 (Figure 3E-K). Because the maize leaf is bilaterally symmetrical, a half-leaf mount rather than a whole-leaf mount may be sufficient for visualization9. To do this, the primordium can be cut with a razor blade along the longitudinal axis after unrolling it up to the midrib, allowing only half of the leaf to be mounted. Another limitation of protocol section 3 is that the thickness of the leaf can limit the optical resolution of the fluorophore signal during deep imaging. To address this issue, it is possible to employ a tissue clearing technique60. However, we found that ClearSee61, a commonly used clearing reagent for imaging plant tissues, is not compatible with the protocol because it causes the sample and coverslip to detach from the nano tape. A potential solution to this problem could be to apply a semipermeable membrane31 over the leaf sample, allowing it to be treated with the clearing solution while being held in place by the nano tape. Such a method that allows liquid solutions to be applied to the unrolled leaf could also be used for whole-mount RNA in situ hybridization and immunolocalization techniques, which have previously been optimized for developing maize inflorescences but not for whole-leaf primordia62,63.
We described protocols for maize, which has large leaf primordia even at the seedling stage. Other grass species with much smaller leaf primordia, such as rice, barley, wheat, Setaria, and Brachypodium16,23,64,65,66, may require the use of additional precision tools to effectively apply these protocols. Furthermore, these protocols were not intended for live cell imaging, which captures real-time dynamic processes of tissue formation and cellular responses. However, as fluorescent probes, imaging technologies, and computing capabilities continue to advance in live cell imaging for plants67, future research could build on these protocols to develop live cell imaging strategies tailored to the unique features of grass leaf primordia.

Grass crops are a major source of food and biofuel for the global population1, and improving leaf anatomy has the potential to increase their productivity2,3. However, our current understanding of how leaf anatomy is regulated in grasses is limited4 and requires the analysis of leaf primordia, as many anatomical and physiological traits of the leaf are predetermined early in development5,6,7. Cellular imaging techniques, such as fluorescence and confocal imaging, are indispensable for studying grass leaf anatomy and cellular traits, but these techniques are difficult to apply to grass leaf primordia because they are deeply ensheathed and rolled within the leaf whorl. We addressed this issue by developing methods for preparing transverse sections and unrolled whole-leaf mounts for fluorescence and confocal analysis of maize leaf primordia, a model system for studying grass leaf anatomy and development2,8.
The maize leaf, like all grass leaves, consists of a strap-like blade with a sheath that wraps around the stem and developing shoot9,10,11,12,13. The leaves develop from the shoot apical meristem (SAM) in a distichous pattern, where each new leaf initiates in the opposite position of the previous leaf, resulting in two ranks of leaves along the vertical axis&#160;(Figure 1A)14 . The developmental stage of each leaf primordium is identified by its position relative to the SAM, with the closest primordium designated as plastochron1 (P1) and the following primordia designated as P2, P3, and so on (Figure 1B,C)2. During development (Figure 1D), the leaf primordium first appears as a crescent-shaped buttress around the base of the SAM (P1), and then grows into a hood-shaped primordium that extends over the meristem (P2)9,10,11. The basal margins of the hood then expand laterally and overlap each other as the tip grows upward, forming a cone-shaped primordium (P3-P5)10. The primordium then rapidly grows in length, and the sheath-blade boundary at the base becomes more prominent with the formation of the ligule, the fringe-like projection on the adaxial side of the leaf (P6/P7). Finally, the leaf unrolls as it emerges from the whorl during steady-state growth, in which the dividing cells are restricted within the small basal region of the blade, forming a gradient with expanding and differentiating cells along the proximal-distal axis (P7/P8)15. The shoot apex of a maize seedling contains multiple primordia at different stages of development, making it an excellent model for studying leaf development8.
Accurate analysis of early leaf development requires staging or the use of standardized criteria to define distinct stages of primordium development in relation to other growth or morphological parameters. Because the leaf primordia are hidden within the grass shoot, investigators typically use parameters such as the age of the plant or the size of the emerging leaves as predictors for the stages and sizes of the leaf primordia9,16. In maize, the chronological age of the plant is determined either by the number of days after planting or germination (DAP/DAG)17,18. The vegetative stage (V stage) is determined by the uppermost leaf with a visible collar, a pale line on the abaxial side between the blade and the sheath that corresponds to the position of the ligule and auricles, a pair of wedge-shaped regions at the base of the blade&#160;(Figure 1A,B)17,19 . Between 20 and 25 DAG, the SAM transitions into an inflorescence meristem and ceases to produce new leaves20. The growth rates of maize leaf primordia can vary depending on the environment and the genotype of the plant. For this reason, plant age and the size of the emerging leaves cannot accurately predict the sizes of leaf primordia; however, using these parameters can help predict the range of primordia stages and sizes for experimental purposes.
Transverse section analysis is a popular method for examining leaf anatomy and development in maize and other grasses because it allows for the sampling of multiple plastochrons in a single section across the shoot21,22,23. This method is also convenient for cellular imaging of fresh samples, as the surrounding leaves serve as a scaffold&#160;that keeps the leaf primordia in place during sectioning and mounting24. However, a disadvantage of this method is that it can be challenging to precisely locate the target plastochron and region within the primordium when sectioning from an intact shoot. Furthermore, because leaf growth varies across plastochrons and along the proximal-distal axis2,5, imprecise sampling could result in an incorrect interpretation of the developmental stage and region of the primordium in a given section. Therefore, developing a method for precise transverse section sampling is critical for ensuring the accuracy and reproducibility of anatomical and developmental analyses of grass leaf primordia.
Whole-leaf mount analysis enables the comprehensive and integrative investigation of tissue and cellular processes that occur at the whole-organ scale, such as proliferative growth25 and vein patterning26,27,28. The method provides a paradermal overview of the leaf, allowing the discovery of distinct processes and patterns that would otherwise be difficult to detect using transverse section analysis24,27. Unlike in Arabidopsis, where there are already established methods for imaging whole-leaf mounts29,30, there is currently no standard method for imaging unrolled whole-leaf mounts in grasses. A previous protocol for unrolling isolated maize leaf primordia involved uncommon materials and was not suitable for cellular imaging31. Advanced imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI), can acquire 3D anatomical information without isolating and unrolling the primordia11,32,33, but they are expensive and require specialized equipment. Developing a technique to overcome the constraints imposed by the rolled and conical morphology of leaf primordia in maize and other grasses would advance investigations into their anatomical and developmental traits.
Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging. We used these methods to quantify vein number and map the spatial-temporal hormone distribution in maize leaf primordia with fluorescent proteins (FPs)24. The first method involves removing the upper portion of older leaves from maize seedlings with a wire stripper (Figure 1E). By exposing the tip of the primordium (P5-P7), it becomes possible to determine its length without having to completely remove the older surrounding leaves, thus enabling easy and accurate sectioning. The second method involves unrolling and mounting whole-leaf primordia (P3-P7) with clear, double-sided nano tape (Figure 1F). These methods are suitable for visualizing various FPs24&#160;but need optimization for using fluorescent dyes and clearing reagents. In addition, we outline some procedures for flattening z-stacks, stitching images, and merging channels in ImageJ/FIJI34, which apply to the images produced by the two methods. These methods are useful for routine fluorescence or confocal imaging of maize leaves, but they can also be adapted for other model grass species, such as rice, Setaria, and Brachypodium.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65239/65239fig01.jpg" /> 
Figure 1: Organization and morphology of maize leaf primordia and overview of the methods. (A) Schematic representation of a maize seedling. Maize has a distichous phyllotaxy, with the new leaf initiating at the opposite position of the previous leaf. The leaf number indicates the chronological order in which the leaves emerged from germination (i.e., first leaf, L1; second leaf, L2; third leaf, L3; etc.). Each leaf has a distal blade and a basal sheath delineated by a collar that corresponds to the ligule and auricle. The uppermost leaf with the collar visible denotes the vegetative stage. The seedling in this example is at the V2 stage, with the L2 collar (arrowhead) visible. The scissors icon indicates the location at the mesocotyl (me) where the seedling must be cut in order to be collected. (B) Schematic representation of the dissected shoot showing isolated L1 to L4, with the leaf primordia L5 to L9 shown as an enlarged image in (C). The plastochron number indicates the position of the primordium relative to the SAM, with the youngest leaf primordium (P1) closest to the SAM and the older leaf primordia (P2, P3, P4, and so on) successively farther away2. (D) Schematic representation of the morphology of maize leaf primordia from P1 to P5. (E) Schematic overview of the method for transverse section analysis of the maize leaf primordia. (1) Trim the older leaves with a wire stripper. (2) Measure the primordium and section the shoot. (3) Mount the section on a slide for imaging and processing (4, 5). (F) Schematic overview of the method for whole-mount analysis of the maize leaf primordia. (1) Remove the surrounding leaves to extract the primordium. (2) Cut and unroll the primordium flat on the nano tape. (3) Mount the sample for imaging and processing (4, 5). Abbreviations: L = leaf; bl = blade; sh = sheath; co = collar; me = mesocotyl; V = vegetative; P = plastochron; SAM = shoot apical meristem. <a href="https://www.jove.com/files/ftp_upload/65239/65239fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Acrylic Gel Clear Double Sided Nano Tape 16.5 ft x 1.2 in, 2 mm thick</td><td>EZlifego Store (Amazon)</td><td>B07YB1ZXG6</td><td>1 roll</td></tr><tr><td>Bellucci Pick Curved micro probe 16.8 cm, 6.6 in</td><td>Bausch &amp;amp; Lomb</td><td>N1692 9</td><td>1 pc</td></tr><tr><td>Clayman guide microprobe Sinskey hook angled shaft, 11.6 cm, 4.6 in</td><td>Storz Opthalmic Instruments</td><td>E0542</td><td>1 pc</td></tr><tr><td>Dental Probe, Bent Needle, 14 cm (5.5 in)</td><td>Ted Pella</td><td>13553</td><td>1 pc</td></tr><tr><td>DOWELL 10-22 AWG Wire Stripper</td><td>Dowell Store (Amazon)</td><td>10-22 AWG</td><td>1 pc</td></tr><tr><td>Feather Double Edge Carbon Steel Blades</td><td>Ted Pella</td><td>121-9</td><td>pkg/10; for fine sectioning</td></tr><tr><td>Frosted End Glass Microscope Slides, 75 mm x 25 mm x 1-1.2 mm</td><td>Ted Pella</td><td>260442</td><td>pkg/144</td></tr><tr><td>GEM Single Edge, Stainless Steel Uncoated Blades</td><td>Ted Pella</td><td>121-1</td><td>box/200; for general cutting/sectioning</td></tr><tr><td>Glycerol</td><td>Thermo Scientific</td><td>PI17904</td><td>1 liter</td></tr><tr><td>ImageJ/FIJI with EDF plugin (version 17.05.2021) and Grid/Collection Stitching plugin</td><td>National Institutes of Health (NIH) USA</td><td>version 2.9.0/1.54s</td><td>The EDF plugin was developed by Alex Prudencio, Jesse Berent, and Daniel Sage for the Biomedical Imaging Group, &amp;Eacute;cole Polytechnique F&amp;eacute;d&amp;eacute;rale de Lausanne (EPFL; http://bigwww.epfl.ch/demo/edf/). The grid/collection stitching software was developed by Stephan Preibisch for the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG).</td></tr><tr><td>Kimwipes Ex-L Small 111.76 mm x 213.36 mm</td><td>Kimtech Science</td><td>34155</td><td>box/280 ply</td></tr><tr><td>Micro Cover Glasses, 22 mm x 22 mm x 0.13 - 0.16 mm thick</td><td>Ted Pella</td><td>260140</td><td>1 ounce</td></tr><tr><td>PU Gel Clear Double Sided Nano Tape 29.5 ft x 1.18 in, 1 mm thick</td><td>Yecaye Store (Amazon)</td><td>L354 W1.18</td><td>2 rolls</td></tr><tr><td>Superslip Cover Glasses, 24 mm x 50 mm x 0.13 - 0.16 mm thick</td><td>Ted Pella</td><td>260166</td><td>1 ounce</td></tr><tr><td>Superslip Cover Glasses, 24 mm x 60 mm x 0.13 - 0.16 mm thick</td><td>Ted Pella</td><td>260168</td><td>1 ounce</td></tr><tr><td>Tempered Glass Cutting Board</td><td>Hacaroa (Amazon)</td><td>B09XMXBT5S</td><td>4 pc</td></tr></tbody></table>

improved methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study early leaf development. Here, we describe methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging. The first method uses a wire stripper to remove the upper portions of older leaves, exposing the tip of the leaf primordium and allowing its measurement for more accurate transverse section sampling. The second method uses clear, double-sided nano tape to unroll and mount whole-leaf primordia for imaging. We show the utility of the two methods in visualizing and analyzing fluorescent protein reporters in maize. These methods provide a solution to the challenges presented by the distinctive morphology of maize leaf primordia and will be useful for visualizing and quantifying leaf anatomical and developmental traits in maize and other grass species.

Transverse section analysis of maize leaf primordia 
We used protocol section 2 to quantify vein number and characterize hormone response patterns in transverse sections of maize leaf primordia with FPs (Figure 4)24. To assess the role of the plant hormone auxin in leaf growth and vein formation, we quantified the number of veins in the leaf primordia of an auxin-deficient maize mutant, vanishing tassel254. In early plastochrons, developing veins exhibit distinct cellularization in the median cell layer of the maize leaf primordium21,22. However, identifying and counting veins using conventional histological techniques22 can be labor-intensive and time-consuming. Thus, to quantify the veins, we utilized the maize auxin efflux protein marker pZmPIN1a::ZmPIN1a:YFP41 (PIN1a-YFP hereafter), which marks developing veins and procambial strands (PCSs; Figure 4A). Using protocol section 2, we were able to standardize the transverse section sampling by measuring the primordia prior to sectioning (Figure 4B,C). We discovered a trend in which vt2 has a wider primordium and more veins than normal (Figure 4D,E)24, which is consistent with data from fully expanded leaves55, indicating that the defect in vt2 began early in leaf development. Using protocol section 2, we were also able to systematically examine the expression patterns of hormone response FP reporters in the leaf primordia (see examples of the images in Figure 4F-I). Through a standardized transverse section sampling scheme, we mapped the distribution of auxin, cytokinin (CK), and gibberellic acid (GA) responses across different plastochrons and regions of leaf primordia, and discovered novel response patterns that we hypothesize have implications for leaf&#160;growth and vein formation24. Therefore, these representative results demonstrate the utility of protocol section 2 for transverse section analysis of maize leaf primordia.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65239/65239fig04.jpg" /> 
Figure 4: Representative results for transverse section analysis of maize leaf primordia. (A-E) Quantification of vein number and primordium width in leaf primordia of normal and vanishing tassel2 with the auxin efflux protein marker PIN1a-YFP. (A) Representative fluorescence images of transverse sections of P5 to P7 expressing PIN1a-YFP in developing veins and procambial strands. The transverse sections were imaged with an epifluorescence microscope using a FITC filter (495-519 nm excitation). The number of veins and the primordium width were quantified using the multi-point and freehand line tools in FIJI/ImageJ, respectively. (B) A maize seedling with the upper leaf whorls removed with a wire stripper to expose the tip of the fourth leaf (L4). The bracket spans the projected primordium length, with the dashed line indicating the mid-length. (C) Schematic diagram of varying primordium shapes from P5 to P7, illustrating how vein number and primordium width at the mid-length (horizontal dashed line) can vary depending on the developmental stage of the primordium. (D,E) Measurement of primordium width (D) and vein number (E) at the mid-length section of the L4 of normal and vt2. The trendline represents 10 mm rolling averages of measurements &#177; standard error of the mean (SEM). (F-I) Representative confocal images of transverse sections of leaf primordia expressing combinations of PIN1a-YFP, auxin response reporter, DR5-RFP, cytokinin response reporter, TCS-Tomato, gibberellic acid-responsive marker, GAR2-YFP, and mDII-Venus, a mutated version of the auxin signaling input reporter DII-Venus. The FP channels are superimposed on the bright-field channel in each image. Scale bar = 200 &#181;m (A); 10 mm (B); 100 &#181;m (F-I). This figure has been modified and reproduced with permission from Robil and McSteen24. Abbreviations: DAG = days after germination; P = plastochron; vt2 = vanishing tassel2; PCS = procambial strand; YFP = yellow fluorescent protein; FITC = fluorescein isothiocyanate; RFP = red fluorescent protein; PIN1a-YFP = pZmPIN1a::ZmPIN1a:YFP; DR5-RFP = DR5rev::mRFPer; TCS-Tomato = TCSv2::NLS-tdTomato; GAR2-YFP = pZmGAR2::ZmGAR2:YFP; mDII-Venus = pZmUbi:mDII:YFP-NLS. <a href="https://www.jove.com/files/ftp_upload/65239/65239fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Whole-mount analysis of maize leaf primordia 
We followed protocol section 3 to visualize and analyze FP expression in whole-leaf mounts of maize leaf primordia (Figure 5). By visualizing vein patterns with PIN1a-YFP, we found that the formation of veins occurs in the entire primordium during early plastochrons, but this process becomes restricted in the proximal regions later in development (Figure 5A)24. Complementary to the transverse section analyses, the whole-leaf mount analyses have revealed tissue- and stage-specific patterns of hormone response during vein formation24. One example is the expression pattern of the CK response reporter TCSv2::NLS-tdTomato37 (TCS-Tomato), relative to PIN1a-YFP expression (Figure 5B)24. By following protocol section 3, we were able to perform both qualitative and quantitative analyses of FP expression in the leaf primordia (Figure 5D-F; unpublished data). We examined the expression patterns of an auxin response reporter, DR5rev::mRFPer41 (DR5-RFP), and a GA-responsive marker, pZmGAR2::ZmGAR2:YFP39 (GAR2-YFP), in whole-leaf mounts of single and double mutants of vt2 and dwarf plant3 (d3), a GA-deficient mutant56 (Figure 5D). We also compared the relative levels of cell proliferation between normal and vt2 leaf primordia using pZmCyclin-D2B::ZmCyclin-D2B:YFP42 (Cyclin-D2B-YFP), which is a marker for the G1/S transition in the cell cycle57 (Figure 5E,F). While there was no significant difference between normal and vt2, Cyclin-D2B-YFP expression was consistent with the known cell proliferation profile of early plastochrons31. We conclude that protocol section 3 is an effective method for analyzing whole mounts of maize leaf primordia, which are difficult to image due to their rolled morphology.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65239/65239fig05.jpg" /> 
Figure 5: Representative results for whole-mount analysis of maize leaf primordia. (A) Representative fluorescence images of leaf primordia of 7 DAG maize seedlings showing developing veins and procambial strands, as marked by the auxin efflux protein marker PIN1a-YFP. P3-P6 primordia were excised from the base, unrolled, and flattened with the adaxial side up. Insets show close-ups of P3 and P4. In P5 and P6, dashed lines demarcate the distal end of the proliferative zones, where the majority of procambial strands are still developing and extending. (B,C) Representative confocal images showing the expression of PIN1a-YFP and cytokinin response reporter, TCS-Tomato, in the proximal marginal region of a P5 primordium. (D) Representative fluorescence images of P4 primordia showing the expression of auxin response reporter, DR5-RFP, and gibberellic acid-responsive marker, GAR2-YFP in normal and single and double mutants of vanishing tassel2 and dwarf plant3. (E) Representative confocal images of P3 and/or P4 showing the expression of Cyclin-D2B-YFP, a reporter for the G1/S transition in the cell cycle. (F) Relative amounts of cell proliferation in P3/P4 leaf primordia of normal and vt2, quantified by measuring the integrated density of the Cyclin-D2B-YFP signal over the area of the primordium using ImageJ/FIJI. The bars represent the mean measurements &#177; standard error of the mean. Scale bar = 500 &#181;m (A,D,E); 200 &#181;m (B); 100 &#181;m (C). Figure 4A-C has been modified and reproduced with permission from Robil and McSteen24, while Figure 4D-F is unpublished data from the authors. Abbreviations: DAG = days after germination; P = plastochron; vt2 = vanishing tassel2; d3 = dwarf plant3; YFP = yellow fluorescent protein; RFP = red fluorescent protein; PIN1a-YFP = pZmPIN1a::ZmPIN1a:YFP; TCS-Tomato = TCSv2::NLS-tdTomato; DR5-RFP = DR5rev::mRFPer; GAR2-YFP = pZmGAR2::ZmGAR2:YFP; Cyclin-D2B-YFP = pZmCyclin-D2B::ZmCyclin-D2B:YFP; ns = no significant difference; au = arbitrary unit. <a href="https://www.jove.com/files/ftp_upload/65239/65239fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Examples of leaf development staging in maize seedlings. <a href="https://www.jove.com/files/ftp_upload/65239/65239_S_File_1_Leaf_staging_maize_seedling_Fall2019 (1).xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Figure S1: Plant samples and materials used in the protocols. (A,B) A maize seedling 7-8 DAG with the upper leaf whorl removed using a wire stripper. (B) The inset shows a close-up of the shoot with the exposed P6 primordium. (C-G) Shoots of maize seedlings with the upper whorls removed for transverse section analysis (C,D) and surrounding leaves fully removed for whole-mount analysis (E-G). (H) A roll of polyurethane gel clear double-sided nano tape. (I,J) P6 leaf primordium (I) and proximal region of P8 leaf (J) unrolled and mounted on glass slides with the nano tape. (K) A glass slide with an unrolled leaf primordium mounted on the stage of an epifluorescence microscope. Abbreviation: DAG = days after germination. <a href="https://www.jove.com/files/ftp_upload/65239/S_FIGURE_1.pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging at JoVE.com

Author Spotlight: Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging  

Maize leaf primordia are deeply ensheathed and rolled, making them difficult to study. Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging.

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study ...

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2.7K Views.  Ateneo de Manila University. Maize leaf primordia are deeply ensheathed and rolled, making them difficult to study. Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging.

Video: Author Spotlight: Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging  

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Biochemistry

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Developmental Biology

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic development. The activity and morphology of SAMs determine important agronomic traits, such as shoot architecture, size and number of reproductive organs, and most importantly, grain yield. Here, we provide a detailed protocol for analyzing both the surface morphology and the internal cellular structure of the living SAMs from different species through laser scanning confocal microscope. The whole procedure from the sample preparation to the acquisition of high resolution three-dimensional (3D) images can be accomplished within as short as 20 minutes. We demonstrate that this protocol is highly efficient for studying not only the inflorescence SAMs of the model species but also the vegetative meristems from different crops, providing a simple but powerful tool to study the organization and development of meristems across different plant species.

This protocol presents how to live image and analyze the shoot apical meristems from different plant species using laser scanning confocal microscopy.

The shoot apical meristem (SAM) functions as a conserved stem cell reservoir and it generates almost all aboveground tissues during the postembryonic ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of plant leaves or vertebrate lungs further difficulties arise from multiple transitions in refractive index between cellular components, between cells and airspaces and between the biological tissue and the rest of the optical system. Moreover, refractive index mismatches lead to attenuation of fluorophore excitation and signal emission in fluorescence microscopy. We describe here the application of the perfluorocarbon, perfluorodecalin (PFD), as an infiltrative imaging medium which optically improves laser scanning confocal microscopy (LSCM) sample imaging at depth, without resorting to damaging increases in laser power and has minimal physiological impact2. We describe the protocol for use of PFD with Arabidopsis thaliana leaf tissue, which is optically complex as a result of its structure (Figure 1). PFD has a number of attributes that make it suitable for this use3. The refractive index of PFD (1.313) is comparable with that of water (1.333) and is closer to that of cytosol (approx. 1.4) than air (1.000). In addition, PFD is readily available, non-fluorescent and is non-toxic. The low surface tension of PFD (19 dynes cm-1) is lower than that of water (72 dynes cm-1) and also below the limit (25 - 30 dyne cm-1) for stomatal penetration4, which allows it to flood the spongy mesophyll airspaces without the application of a potentially destructive vacuum or surfactant. Finally and crucially, PFD has a great capacity for dissolving CO2 and O2, which allows gas exchange to be maintained in the flooded tissue, thus minimizing the physiological impact on the sample. These properties have been used in various applications which include partial liquid breathing and lung inflation5,6, surgery7, artificial blood8, oxygenation of growth media9, and studies of ice crystal formation in plants10. Currently, it is common to mount tissue in water or aqueous buffer for live confocal imaging. We consider that the use of PFD as a mounting medium represents an improvement on existing practice and allows the simple preparation of live whole leaf samples for imaging.

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

We describe the use of perfluorodecalin as an infiltrative mounting medium. This is a simple method for improving depth of imaging in Arabidopsis thaliana leaf tissue with minimal physiological impact.

The problem of acquiring high-resolution images deep into biological samples is widely acknowledged1. In air-filled tissue such as the spongy mesophyll of ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in ...

Neuroscience

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

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Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Live Confocal Imaging of Developing Arabidopsis Flowers

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Confocal Live Imaging of Shoot Apical Meristems from Different Plant Species

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Upright Imaging of Drosophila Embryos

A Simple Method for Imaging Arabidopsis Leaves Using Perfluorodecalin as an Infiltrative Imaging Medium

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds