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.remotexs.ntu.edu.sg/files/ftp_upload/65239/65239fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; 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, &#201;cole Polytechnique F&#233;d&#233;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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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  

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in biology. Transitional methods require a substantial amount of time and effort to obtain a 3D image and including sectioning the intact tissue, immunolabeling, and imaging serially-sectioned tissue, which produces a loss of information at each step of the process. In recently developed approaches for high-resolution imaging within intact tissue, molecular characterization has been restricted to the labeling of proteins. However, currently available protocols for organ clearing require a considerably long process time, making it difficult to implement tissue clearing techniques in the lab. We recently established a rapid and highly-reproducible protocol termed ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs), which allows for tissue clearance within several hours. Moreover, ACT-PRESTO enables rapid immunolabeling with conventional methods and accelerates antibody penetration into the deep layer of densely-formed, thick specimens by applying pressure or convection flow. We describe how to prepare tissues, how to clear by lipid removal using electrophoresis, and how to immuno-stain by a pressure-assisted delivery. The rapidity and consistency of the protocol will expedite the performance of 3D histological research and volume-based diagnoses.

ACT-PRESTO (active clarity technique&#8211;pressure related efficient and stable transfer of macromolecules into organs) enables rapid, efficient, but inexpensive tissue clearing and rapid antibody penetration into cleared tissues by passive diffusion or pressure-assisted delivery. Using this method, a wide range of tissues can be cleared, immunolabeled by multiple antibodies, and volume-imaged.

The identification and exploration of the detailed organization of organs or of the whole body at the cellular level are fundamental challenges in ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing the innate cellular fluorescence signature from each individual live cell in a population distributed in a three-dimensional (3D) space. The innate fluorescence signature of a cell is a collection of fluorescence signals emitted by various biomolecules within the cell. Previous studies established that innate fluorescence signatures reflect various cellular properties and differences in physiological status and are a rich source of information for cell characterization and identification. Innate fluorescence signatures have been traditionally analyzed at the population level, necessitating a clonal culture, but not at the single-cell level. CRIF is particularly suitable for studies that require 3D resolution and/or selective extraction of fluorescence signals from individual cells. Because the fluorescence signature is an innate property of a cell, CRIF is also suitable for tag-free prediction of the type and/or physiological status of intact and single cells. This method may be a powerful tool for streamlined cell analysis, where the phenotype of each single cell in a heterogenous population can be directly assessed by its autofluorescence signature under a microscope without cell tagging.

Here, a protocol is presented for optically extracting and cataloging innate cellular fluorescence signatures (i.e., cellular autofluorescence) from every individual live cell distributed in a three-dimensional space. This method is suitable for studying the innate fluorescence signature of diverse biological systems at a single-cell resolution, including cells from bacteria, fungi, yeasts, plants, and animals.

Described here is confocal reflection microscopy-assisted single-cell innate fluorescence analysis (CRIF), a minimally invasive method for reconstructing ...

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

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Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Preparing Individual Drosophila Egg Chambers for Live Imaging

Rapid Analysis and Exploration of Fluorescence Microscopy Images

ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Reconstruction of Single-Cell Innate Fluorescence Signatures by Confocal Microscopy