This work was supported by the Japan Science and Technology Agency [PRESTO117665 to S.B., ERATOJPMJER1502 to N.N.] and by the Japan Society for the Promotion of Science (Grants-in-Aid for Research Activity Start-up [22880008 to S.B.] and for Young Scientists (B) [23780040 to S.B.]). We thank A. Senzaki, Y. Suzuki, Y. Sugisawa and E. Betsuyaku for excellent technical assistance, J. Parker for providing the Pst_a2 strain, and T. Demura for providing the pBGYN vector.

NOTE: It is important to prevent sleep movement of living leaf samples during time-lapse imaging. To minimize mechanical stress on leaves, gentle fixation of leaves is necessary. Only adequately prepared leaf samples produce time-lapse images suitable for various image analyses. A protocol using transgenic Arabidopsis plants expressing YFP-NLS fusion under the control of PR1 gene promoter (pPR1-YFP-NLS plants) and Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) is described below as an example.
1. Preparation of plants and pathogens
<ol>
	<li>Fill a plastic cell plug tray (Table of Materials) with autoclaved soil.</li>
	<li>Sow one transgenic Arabidopsis seed per cell (Figure 1A).</li>
	<li>Transfer the tray to a growth room maintained at 23 &#176;C and grow the plants under continuous white light for 2-3 weeks.</li>
	<li>Two days prior to pathogen inoculation, streak Pseudomonas syringae pv. tomato DC3000 avrRpt2 strain (Pst_a2) from a glycerol stock onto NYG medium (5 g/L peptone, 3 g/L yeast extract, 20 mL/L glycerol, 15 g/L bacteriological agar, pH 7.0) containing 100 mg/L rifampicin and 50 mg/L kanamycin and incubate at 28 &#176;C for 48 h.</li>
	<li>Harvest the bacterial cells that appear on the surface of the medium using plastic tips, transfer them to a plastic tube containing 10 mM MgCl2, and resuspend. Measure the optical density (OD) of the solution at 600 nm (OD600). Adjust the final cell concentration of bacterial cells to 108 colony formation units (CFU)/mL, which normally corresponds to OD600 = 0.211.</li>
</ol>
2. Pathogen inoculation
<ol>
	<li>Carefully cut out a cell plug containing a 2-3-week-old plant without damaging the plant. Set the cell in an empty cell plug tray (2 x 2 cells are sufficient) to maintain a good balance (Figure 1B).</li>
	<li>Select a visibly healthy leaf for inoculation. Generally, the third, fourth, and fifth leaves (#3, #4, and #5, respectively, in Figure 1C) from the bottom of the plant are easy to handle. Use leaves at the same position in a set of experiments for better reproducibility. Water the soil holding the plant before inoculation for long-term time-lapse imaging.</li>
	<li>Optionally, in the case of analyzing stress-responsive promoters such as pPR1, to ensure that plants are not naturally stressed, examine the leaves under a fluorescence stereomicroscope prior to pathogen inoculation to verify the absence of YFP signal. Exclude leaves showing YFP signal from the experiment.</li>
	<li>Wear disposable latex gloves before infiltration to avoid direct contact with the pathogen. Using a 1 mL needleless plastic syringe, carefully infiltrate the abaxial side of the leaf with the bacterial suspension (108 CFU/mL)11 (Figure 1D). Inoculation of a small portion on one-half of the leaf enables a good visualization of pPR1 activity; the infiltrated area becomes visible as darker green in color compared with the remaining leaf. Be extremely careful not to cause any mechanical damage to the leaf during infiltration. 
	NOTE: Make sure that all the intercellular spaces in the infiltrated area are completely (vertically) fulfilled with the pathogen suspension; otherwise, the PCD domain will be difficult to visualize under the fluorescent stereomicroscope. This can be simply confirmed by completion of dark greening in the infiltrated area.</li>
	<li>Absorb the excess of bacterial suspension from the area surrounding the infiltrated section of the infiltrated leaf with a soft paper towel.</li>
</ol>
3. Mounting the inoculated leaf
<ol>
	<li>Immediately after inoculation, fix a glass slide on the plastic tray using surgical tape (Table of Materials) such that the infiltrated leaf is located at the center of the glass slide. Ensure that the inoculated leaf blade is completely fitted within the glass slide (Figure 2A).</li>
	<li>Prepare double-layered plastic tape (in the case of 0.2 mm thick tape, see Table of Materials) and cut it into two pieces (Figure 2B; pieces 1 and 2) to fit the spaces along the petiole of the infiltrated leaf. Arrange the length of these two pieces, indicated with a double-headed arrow in Figure 2B, to fit into the length of the double-headed arrow shown in Figure 2A. 
	NOTE: Cutting out a corner from each of the two pieces helps avoid damage to the leaf blade (Figure 2B, arrowheads; also see step 3.4 below). Any kind of plastic tape with similar thickness is suitable for making a bridge over the petiole. The stiffness of plastic tape is important for easy handling during the procedure described below.</li>
	<li>Using a pair of fine tweezers, stick tape pieces 1 and 2 on either side of the petiole such that the cut corners of each piece align with the base of the leaf blade (Figure 2C). Ensure that the tape pieces do not touch the petiole or leaf blade. 
	NOTE: These double-layered plastic tape pieces at the base of the leaf blade act as spacers and prevent physical stress on the petiole during mounting.</li>
	<li>Prepare an additional piece of double-layered plastic tape (Figure 2B, piece 3) to fit the size of the double-headed arrow in Figure 2B and stick it on top of the tape pieces 1 and 2 to form a bridge over the petiole (Figure 2D,E). Be extremely careful not to catch the petiole and leaf blade directly between the tape pieces at positions indicated with arrows in Figure 2D,E.</li>
	<li>Gently stick a small piece of surgical tape (Figure 2F, piece 4) on the glass slide above the tip of the leaf blade so that the leaf blade is fixed very softly on the glass slide. Only firmly press down the part of surgical tape directly touching the glass slide (Figure 2F, area outlined with a dashed red line), not the other part overlying the leaf.</li>
	<li>Gently stick another small piece of surgical tape (Figure 2G, piece 5) at the border of the petiole and plastic tape pieces (1, 2, and 3) so that the petiole is very softly fixed onto both the glass slide and plastic tape pieces. Only firmly attach the part of surgical tape directly touching the glass slide to the glass slide and plastic tape pieces (Figure 2G, area outlined with a dashed red line), not the other part overlying the petiole.</li>
	<li>Prevent neighboring leaves from moving into the field of view of the microscope using 200 &#181;L pipette tips (Figure 2H). Insert the pipette tips in the soil to gently hold the neighboring leaves away from the infiltrated leaf. Be careful not to insert the tips too deep in the soil to avoid possible root damage. 
	NOTE: The prepared plant is now ready for fluorescence stereomicroscope imaging.</li>
</ol>
4. Microscopic time-lapse observation
<ol>
	<li>Turn on the fluorescent stereomicroscope. 
	NOTE: Here, an automated stereomicroscope equipped with a highly sensitive 1.4 megapixel monochrome digital camera in 12-bit mode is used (Table of Materials). The microscope should be placed in a dark room fitted with an air-conditioning unit or in a dark cabinet with adequate ventilation to maintain the room temperature at 23 &#176;C during time-lapse imaging.</li>
	<li>Set the plant in the space above under the objective lens of the stereomicroscope for imaging.</li>
	<li>Set up the parameters for time-lapse imaging (see examples in Table 1). Make sure to program steps for light exposure during the interval period of time-lapse imaging since light has a major impact on plant immunity12.
	<ol>
		<li>Use the conventional YFP filter (excitation 500-520 nm; emission 540-580 nm) to visualize the YFP signal.</li>
		<li>Use the Texas Red (TXR) filter (excitation 540-580 nm; emission 610 nm long pass) to visualize chlorophyll autofluorescence so that the PCD domain is visible as a dark area (no autofluorescence) surrounded by YFP-positive cell layers9 (Figure 3A).</li>
		<li>Use the conventional epi-bright field setup for additional light exposure steps. 
		NOTE: Before the experiment, measure the power of epi-bright light source and adjust it to the level of own plant growth condition.</li>
	</ol>
	</li>
	<li>Run the time-lapse imaging program. For long-term observation over several days, consider watering the plant appropriately, for example, during the light exposure steps.</li>
	<li>After image acquisition, omit extra channels used for light exposure in the intervals (corresponding to channels 3-8 in Table 1) from the data set. Analyze data with various methods, such as region-of-interest (ROI) analysis, using different image analysis software such as Fiji9.</li>
</ol>

<ol>
	<li>Tsuda, K., Somssich, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+networks+in+plant+immunity.">Transcriptional networks in plant immunity.</a> New Phytologist. 206 (3), 932-947 (2015).</li><li>Mine, A., Sato, M., Tsuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+systems+understanding+of+plant-microbe+interactions.">Toward a systems understanding of plant-microbe interactions.</a> Frontiers in Plant Science. 5, (2014).</li><li>Murray, S. L., Thomson, C., Chini, A., Read, N. D., Loake, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel,+defense-related+Arabidopsis+mutant,+cir1,+isolated+by+luciferase+imaging.">Characterization of a novel, defense-related Arabidopsis mutant, cir1, isolated by luciferase imaging.</a> Molecular Plant-Microbe Interactions. 15 (6), 557-566 (2002).</li><li>Spoel, S. H., Johnson, J. S., Dong, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+tradeoffs+between+plant+defenses+against+pathogens+with+different+lifestyles.">Regulation of tradeoffs between plant defenses against pathogens with different lifestyles.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (47), 18842-18847 (2007).</li><li>Asai, S., Shirasu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+cells+under+siege:+plant+immune+system+versus+pathogen+effectors.">Plant cells under siege: plant immune system versus pathogen effectors.</a> Current Opinion in Plant Biology. 28, 1-8 (2015).</li><li>Schmelzer, E., Kruger-Lebus, S., Hahlbrock, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+Spatial+Patterns+of+Gene+Expression+around+Sites+of+Attempted+Fungal+Infection+in+Parsley+Leaves.">Temporal and Spatial Patterns of Gene Expression around Sites of Attempted Fungal Infection in Parsley Leaves.</a> The Plant Cell. 1 (10), 993-1001 (1989).</li><li>Ohshima, M., Itoh, H., Matsuoka, M., Murakami, T., Ohashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+stress-induced+or+salicylic+acid-induced+expression+of+the+pathogenesis-related+1a+protein+gene+in+transgenic+tobacco.">Analysis of stress-induced or salicylic acid-induced expression of the pathogenesis-related 1a protein gene in transgenic tobacco.</a> The Plant Cell. 2 (2), 95-106 (1990).</li><li>Rushton, P. J., Reinst&#228;dler, A., Lipka, V., Lippok, B., Somssich, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+plant+promoters+containing+defined+regulatory+elements+provide+novel+insights+into+pathogen-+and+wound-induced+signaling.">Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling.</a> The Plant Cell. 14 (4), 749-762 (2002).</li><li>Betsuyaku, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salicylic+Acid+and+Jasmonic+Acid+Pathways+are+Activated+in+Spatially+Different+Domains+Around+the+Infection+Site+During+Effector-Triggered+Immunity+in+Arabidopsis+thaliana.">Salicylic Acid and Jasmonic Acid Pathways are Activated in Spatially Different Domains Around the Infection Site During Effector-Triggered Immunity in Arabidopsis thaliana.</a> Plant &amp; Cell Physiology. 59 (1), 8-16 (2018).</li><li>Dodds, P. N., Rathjen, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+immunity:+towards+an+integrated+view+of+plant-pathogen+interactions.">Plant immunity: towards an integrated view of plant-pathogen interactions.</a> Nature Reviews Genetics. 11 (8), 539-548 (2010).</li><li>Katagiri, F., Thilmony, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> Arabidopsis Book. , (2002).</li><li>Zeier, J., Pink, B., Mueller, M. 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The authors have nothing to disclose.

Here, we report a simple method to mount a living Arabidopsis leaf expressing a fluorescent reporter gene under the control of a promoter of interest for long-term observation using an automated fluorescent stereomicroscope. Time-lapse imaging of a fluorescent reporter has been frequently performed in root tissues; however, only a few similar studies have been conducted in leaf tissues. This is most likely because leaves are able to freely move in space, whereas roots are often buried and fixed in solid agar medium.
In this report, we focused on the spatiotemporal dynamics of pPR1 activity during ETI induced by Pst_a2. In addition to the gentle fixation of the leaf detailed above, it is important to clearly visualize the spatiotemporal dynamics of cellular events such as promoter activation and PCD. If the distinction between cells showing pPR1 activity and PCD is not sharp, make sure that all of the intercellular spaces in the infiltrated area are completely filled with the pathogen suspension (see step 2.4). This is critical when using wide-field fluorescence stereomicroscopes since these microscopes capture all detectable signals along the same vertical position of the specimen. Chlorophyll autofluorescence from surviving cells above or beneath the cells in the PCD domain easily masks the dead cells exhibiting no autofluorescence. This is also true for YFP signal.
Conditions for time-lapse imaging need to be established carefully through several preliminary experiments under different experimental conditions. Parameters for time-lapse imaging depend on several factors such as the microscopic system, transgenic plants, and pathogens. To obtain these parameters, we first analyzed various exposure times for YFP signal intensity in the infiltrated leaf at 7 hpi, which almost coincides with the initial activation of pPR1. A 5 s exposure was determined as appropriate for capturing YFP signal with the stereomicroscope used in this study. A similar test was performed for imaging chlorophyll autofluorescence. Exposure of the specimen to light between 3 min intervals was programmed into the time-lapse imaging program as normal bright field imaging with maximum exposure time. Our system (Table of Materials) allowed us to have 2.5 min in addition to YFP, TXR, and bright field imaging. This constraint was the primary reason for choosing a 3 min interval. Next, we confirmed that this time-lapse condition caused no apparent damage to the plant samples, and did not induce ectopic light stress-related activation of pPR1 (Figure 3B,C). This led to the development of the program used in this study. Thus, 3-min intervals of fluorescence imaging were deemed sufficient for capturing pPR1 dynamics during Pst_a2-mediated ETI9.
Promoter-reporter constructs, especially with the fluorescent reporter fused to the NLS, have been utilized by many groups, and are easily available from the research community; we used the construct published by Kubo&#160;et al.13. Thus, the protocol described here can be used in any plant biology study examining leaf tissues, if appropriate transgenic plants are available. Our simple and easy protocol provides a great opportunity for researchers who are keen to analyze the spatiotemporal dynamics of any biological event occurring in leaves, such as immune response. It is plausible that our method using tape pieces induces a slight physical stress on the specimens. However, this issue can be controlled by including appropriate positive and negative controls, such as mock treatments, in the experiments (Figure 3C). The experimental conditions can be further modified and optimized by analyzing these controls under different conditions.
In recent years, rapid development of imaging instruments and techniques has stimulated the interest of researchers in the complex spatiotemporal aspects of biological events. In any imaging analysis, appropriate mounting and fixing of specimens are among the most important issues. The simple and versatile method of mounting living Arabidopsis leaves developed in this study can be applied and optimized for various imaging experiments.

Plant immune response involves a dynamic transcriptional reprogramming regulated by multiple transcription factors as well as phytohormones1. The accumulation of transcriptome data provides opportunities for collecting information on the plant immune system: for example, the network structure of signaling cascades2. However, our knowledge of the spatial and temporal dynamism of plant immunity still remains limited3,4,5.
In previous studies, spatiotemporal regulation of defense-related gene expression has been mostly analyzed using in situ hybridization and a &#946;-glucuronidase (GUS) reporter assay6,7,8. These methods enable us to visualize the transcriptional activation of various genes of interest in situ. However, these procedures require chemical fixation of specimens, and thus result in the loss of all temporal information. Biological events, such as immunity, progress over time. The use of luciferase as a reporter has enabled the capture of temporal dynamics of the promoter of interest3. However, luciferase-based assay requires an expensive substrate and highly sensitive detectors. To increase our understanding of the spatiotemporal aspects of plant immune response using a simple procedure, we generated transgenic Arabidopsis thaliana plants expressing the yellow fluorescent protein (YFP) gene fused to the nuclear localization signal (YFP-NLS) under the control of the promoter of the defense-related marker gene, Pathogenesis-Related 1 (PR1)9. We used chlorophyll autofluorescence, a marker of living cells, to capture the process of programmed cell death (PCD), which often occurs during effector-triggered immunity (ETI), a form of plant immunity induced by specific pathogen infection9,10. Monitoring the temporal dynamics of fluorescence signal intensity in freely moving objects, such as living Arabidopsis leaves, requires a complex image processing software either built in-house or available commercially. Alternatively, preventing specimens from moving is a simple method to solve the issue. Here, we developed a versatile and simple method for mounting living leaves of a transgenic Arabidopsis plant for long-term observation under an automated fluorescence stereomicroscope. The method allows us to capture the promoter dynamics within soil-grown intact plant leaves over a few days.

<table border="0" cellpadding="0" cellspacing="0" style="width:616px;" width="616">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Agar</td>
			<td style="width:115px;">BD biosciences</td>
			<td style="width:119px;">214010</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Protease Peptone No. 3</td>
			<td style="width:115px;">BD biosciences</td>
			<td style="width:119px;">211693</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bacto Yeast Extract</td>
			<td style="width:115px;">BD biosciences</td>
			<td style="width:119px;">212750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BM2 soil</td>
			<td style="width:115px;">Berger</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:115px;">nacalai tesque</td>
			<td style="width:119px;">17017-35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kanamycin sulfate</td>
			<td style="width:115px;">Wako</td>
			<td style="width:119px;">113-00343</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Leica M205FA with DFC365FX, LED_MCI and Leica EL6000 (Las X software-regulated)</td>
			<td style="width:115px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Yellow fluorescent protein (YFP) and Texas Red (TXR) filters installed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Magnesium Chloride Hexahydrate</td>
			<td style="width:115px;">Wako</td>
			<td style="width:119px;">135-00165</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Micro Slide Glass (Size: 76 mm &#215; 26 mm, Thickness: 1.0&#8211;1.2 mm)</td>
			<td style="width:115px;">MATSUNAMI</td>
			<td style="width:119px;">S1112</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropore Surgical Tape (1.25 cm &#215; 9 m)</td>
			<td style="width:115px;">3M</td>
			<td style="width:119px;">1530-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needleless 1 ml plastic syringe</td>
			<td style="width:115px;">Terumo</td>
			<td style="width:119px;">SS-01T</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Plastic cell plug tray (cell size: 44 mm&#160; &#215; 44mm &#215; 44 mm)</td>
			<td style="width:115px;">Tanaka sangyo, Japan</td>
			<td style="width:119px;">htray-bk72</td>
			<td style="width:167px;">Any&#160; tray with similar sized cells can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rifampicin</td>
			<td style="width:115px;">nacalai tesque</td>
			<td style="width:119px;">30259-81</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Plastic Tape (0.2 mm thick &#215; 19 mm wide &#215; 10 m long)</td>
			<td style="width:115px;">Yamato</td>
			<td style="width:119px;">No0200-19-1</td>
			<td style="width:167px;">Any plastic/vinyl tape with&#160; similar thickness can be used</td>
		</tr>
	</tbody>
</table>

a versatile method for mounting arabidopsis leaves for intravital time-lapse imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

Here, we used Pst_a2-induced ETI as an example for time-lapse imaging. Time-lapse data were obtained as a series of images, a few of which are shown in Figure 3B, and as a time-lapse movie (Supplemental Movie 1)9. In successful experiments using pPR1-YFP-NLS during Pst_a2-induced ETI, transient activation of pPR1 was observed, as evident from YFP expressing foci, in several layers of cells surrounding the PCD domain (Figure 3B)9. The activation of pPR1 in cells surrounding the PCD domain usually starts at approximately 5 hours post inoculation (hpi), peaks at approximately 12 hpi, and lasts up to 40 hpi (Figure 3B)9. Since images were acquired using an epi-fluorescent system, the YFP signals obtained here were generated from several adaxial cell layers, including epidermal as well as upper mesophyll cells.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59147/59147fig1.jpg" /> 
Figure 1: Method used for infiltration of bacterial pathogen into the Arabidopsis&#160;thaliana leaf. (A) Two- to three-week-old Arabidopsis plants were grown in a cell plug tray. (B) One-cell plug containing the selected plant to be used for inoculation and observation was cut out and placed into the empty cell plug tray to maintain balance. (C)The third, fourth, and fifth leaves (#3, #4, and #5, respectively) are suitable for image analysis. (D) Infiltration of the pathogen suspension into a selected leaf using a needleless syringe. The infiltrated area can be recognized based on its darker green color than the remaining leaf. <a href="https://www.jove.com/files/ftp_upload/59147/59147fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59147/59147fig2.jpg" /> 
Figure 2: Method used for mounting the infiltrated Arabidopsis leaf for time-lapse imaging. (A)&#160;Photograph of the Arabidopsis plant with a glass slide fixed under the inoculated leaf. The infiltrated leaf was positioned at the center of the glass slide. The double-headed arrow indicates the length of the plastic tape spacers. (B) Preparation of plastic tape spacers and bridge. Double -layered plastic tape was cut into two pieces (1 and 2) for the spacers, and another piece (3, outlined with a dashed red line) was cut to prepare a bridge. The lengths of double-headed arrows are almost identical to the length of the double-headed arrow in (A). One of the four corners of pieces 1 and 2, indicated with arrows, were cut. (C) Two pieces of double-layered plastic tape (numbered as 1 and 2) were carefully pasted onto the slide glass along the petiole using a pair of fine tweezers, without making direct contact with the petiole and leaf blade. (D) An additional piece of double-layered plastic tape (numbered as 3), which was prepared from (B), was placed on both basal spacers (1 and 2) to form a bridge over the petiole, while ensuring not to catch the petiole between tapes 1 and 2 at positions indicated with arrows. (E) Photograph showing the taped petiole. It is important not to catch plant tissue directly between the plastic tape pieces at positions indicated with arrows. (F) A small piece of surgical tape (numbered as 4) was pasted gently on the glass slide around the tip of the leaf blade. Only the area outlined by the dashed red line was pressed firmly onto the glass slide. (G) Another small piece of surgical tape (numbered as 5) was gently pasted at the border of the petiole and plastic tape pieces. Only the area outlined by the dashed red line was pressed for fixing the petiole softly on the slide glass and plastic tape pieces. (H) Two disposable pipette tips were used to prevent neighboring leaves from moving into the field of view of the stereomicroscope. The pipette tips were inserted directly into the soil at appropriate positions. <a href="https://www.jove.com/files/ftp_upload/59147/59147fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59147/59147fig3.jpg" /> 
Figure 3: Representative results using the Arabidopsis leaf mounting method. (A) Fluorescent stereomicroscopic images of a transgenic Arabidopsis leaf (pPR1-YFP-NLS plant). A portion of the leaf was infiltrated with Pst_a2 (108 CFU/mL) on its abaxial surface, and images were captured at 22 hours post inoculation (hpi). Nuclei in which the pPR1 promoter was activated were detected using the YFP filter, and programmed cell death (PCD) was detected based on the loss of chlorophyll autofluorescence using the Texas Red (TXR) filter. Scale bar = 2.5 mm. (B) A few time-lapse images selected from a collection of 800 images obtained using the protocol described here. These data provide an in vivo overview of the spatiotemporal dynamics of pPR1 activity for 40 hpi. Merged images of YFP and TXR images are shown. Scale bar = 2.5 mm. (C) Mock infiltration of a transgenic leaf (pPR1-YFP-NLS plant). In mock infiltration, 10 mM MgCl2 was infiltrated into the abaxial side of the leaf, followed by time-lapse imaging. Mock treatment did not cause ectopic pPR1 activity and did not interfere with chlorophyll autofluorescence at 13 hpi. An arrow indicates the position of mock infiltration. Scale bar = 2.5 mm. These images were modified from a previous study9. <a href="https://www.jove.com/files/ftp_upload/59147/59147fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/59147/59147table1.jpg" /> 
Table 1:Time-lapse imaging program used in this study. 
<img alt="Supplemental Movie 1" src="/files/ftp_upload/59147/59147movie1.jpg" /> 
Supplemental Movie 1: Time-lapse movie showing the spatiotemporal dynamics of pPR1 activation in a transgenic Arabidopsis leaf following effector-triggered immunity (ETI) induced by Pst_a2&#160;inoculation. A portion of the abaxial side of a transgenic leaf carrying the pPR1-YFP-NLS construct was infiltrated with Pst_a2 (108 CFU/mL). Images were acquired at 3-min intervals for a total of 40 h. The time stamp shown is in the format dd:hh:mm:ss.sss. This movie was modified from a previous study9. <a href="https://www.jove.com/files/ftp_upload/59147/Movie.mov" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging at JoVE.com

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

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8.1K Views.  Japan Science and Technology Agency (JST), PRESTO. We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

Video: A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

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 ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Specific Labeling of Mitochondrial Nucleoids for Time-lapse Structured Illumination Microscopy

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and several essential mitochondrial polypeptides. Mitochondrial nucleoids divide and distribute within the dynamic mitochondrial network that undergoes fission/fusion and other morphological changes. High resolution live fluorescence microscopy is a straightforward technique to characterize a nucleoid&#39;s position and motion. For this technique, nucleoids are commonly labeled through fluorescent tags of their protein components, namely transcription factor a (TFAM). However, this strategy needs overexpression of a fluorescent protein-tagged construct, which may cause artifacts (reported for TFAM), and is not feasible in many cases. Organic DNA-binding dyes do not have these disadvantages. However, they always show staining of both nuclear and mitochondrial DNAs, thus lacking specificity to mitochondrial nucleoids. By taking into account the physico-chemical properties of such dyes, we selected a nucleic acid gel stain (SYBR Gold) and achieved preferential labeling of mitochondrial nucleoids in live cells. Properties of the dye, particularly its high brightness upon binding to DNA, permit subsequent quantification of mitochondrial nucleoid motion using time series of super-resolution structured illumination images.

The protocol describes specific labeling of mitochondrial nucleoids with a commercially available DNA gel stain, acquisition of time lapse series of live labeled cells by super-resolution structured illumination microscopy (SR-SIM), and automatic tracking of nucleoid motion.

Mitochondrial nucleoids are compact particles formed by mitochondrial DNA molecules coated with proteins. Mitochondrial DNA encodes tRNAs, rRNAs, and ...

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because the larval body wall and most internal organs are transparent. However, continuous live imaging of intact Drosophila larvae for longer than 30 min has been challenging because it is difficult to noninvasively immobilizeimmobilizing larvae for a long time. Here we present a larval mounting method called LarvaSPA that allows for continuous imaging of live Drosophila larvae with high temporal and spatial resolution for longer than 10 hours. This method involves partially attaching larvae to the coverslip using a UV-reactive glue and additionally restraining larval movement using a polydimethylsiloxane (PDMS) block. This method is compatible with larvae at developmental stages from second instar to wandering third instar. We demonstrate applications of this method in studying dynamic processes of Drosophila somatosensory neurons, including dendrite growth and injury-induced dendrite degeneration. This method can also be applied to study many other cellular processes that happen near the larval body wall.

LarvaSPA, A Method for Mounting Drosophila Larva for Long-Term Time-Lapse Imaging

This protocol describes a method for mounting Drosophila larvae to achieve longer than 10 h of uninterrupted time-lapse imaging in intact live animals. This method can be used to image many biological processes close to the larval body wall.

Live imaging is a valuable approach for investigating cell biology questions. The Drosophila larva is particularly suited for in vivo live imaging because ...

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.

We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species.

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations ...

Longitudinal Intravital Microscopy Using a Mammary Imaging Window with Replaceable Lid

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in combination with skin flap surgery or implantation of imaging windows has been used to study the dynamics of the healthy mammary gland at different developmental stages. Most mammary imaging technologies are limited to a time frame of hours to days, whereas the majority of mammary gland remodeling processes occur in time frames of days to weeks. To study mammary gland remodeling, methods that allow optical access to the tissue of interest for extended time frames are required. Here, an improved version of the titanium mammary imaging window with a replaceable lid (R.MIW) is described that allows high-resolution imaging of the mammary gland with a cellular resolution for up to several weeks. Importantly, the R.MIW provides tissue access over the entire duration of the intravital imaging experiment and could therefore be used for local tissue manipulation, labeling, drug administration, or image-guided microdissection. Taken together, the R.MIW enables high-resolution characterization of the cellular dynamics during mammary gland development, homeostasis, and disease.

This protocol describes a novel mammary imaging window with a replaceable lid (R.MIW). Intravital microscopy after implantation of the R.MIW allows for longitudinal and multi-day imaging of the healthy and diseased mammary gland with a cellular resolution during the different developmental stages.

The branched structure of the mammary gland is highly dynamic and undergoes several phases of growth and remodeling after birth. Intravital microscopy in ...