We thank Barbara Jakli&#269; for&#160;technical assistance. This research was financially supported by the Slovenian Research and Innovation Agency (research core funding no. P4-0165 and project Z4-3217: Deciphering redox-related signaling interconnectedness in potato resistance against viruses).

NOTE: Sections 1 and 2 describe a modified protocol for plant material preparation based on the methods outlined by Lukan et al.7. Specifically, some modifications to controlled environmental conditions and inoculum preparation were made.
1. Growing potato plants
<ol>
	<li>Grow healthy potato cv. Rywal plants in stem node tissue culture8.</li>
	<li>After 6-8 weeks, cut ten 1 cm-long explants containing nodes from potato plants on sterile paper under sterile conditions using sterile tweezers and a scalpel knife.</li>
	<li>Transfer them to plastic boxes with Murashige and Skoog (MS30) medium (Figure 1A) and grow them under controlled environmental conditions (22 &#176;C in the light and 19 &#176;C in the dark with 250 &#181;E or &#181;mol/m2/s&#160;radiation and a 16-h photoperiod, at a relative humidity of 55% &#177; 5%).</li>
	<li>Transfer them to the soil 2 weeks after micropropagation.
	<ol>
		<li>Fill the pots (r = 10 cm) with soil. Use a finger to create a 3-4 cm deep hole in the soil in the middle of a pot, fill the hole with water, and wait for it to absorb.</li>
		<li>Place one plant per pot into the hole, leaving the leaves above the surface. Gently cover the roots with soil (Figure 1B). 
		NOTE: Some plants might not develop roots. Exclude those plants from the analysis.</li>
	</ol>
	</li>
	<li>Grow the plants in soil in a growth chamber under controlled environmental conditions (22 &#176;C in the light and 19 &#176;C in the dark, at a relative humidity of 55% &#177; 5%, with 250 &#181;mol/m2/s&#160;radiation and a 16-h photoperiod).</li>
	<li>After 3-4 weeks, plants are ready. Use these plants to be inoculated. 
	NOTE: Plants should have at least 3-4 fully developed leaves with visible leaflets (Figure 1C). They should look healthy, with no visible symptoms (yellow or brown leaves), which could be mistaken for lesions caused by PVY. To achieve comparable results, plants should be inoculated at the same time (e.g., at 9 am) in all experiments to avoid possible effects of circadian rhythm on plant immune response and lesion formation9,10,11.</li>
</ol>
2. Inoculum preparation and potato inoculation
<ol>
	<li>Inoculate the plant with PVY strain N-Wilga&#160;(PVYN-Wi; accession no. EF558545) inoculum as stated below. 
	NOTE: More plants can be inoculated in parallel if more than one digital microscope is available. Plants should have at least three fully developed leaves before inoculation (Figure 1C). PVYN-Wi is multiplied and maintained in potato cv. Pentland.
	<ol>
		<li>Prepare phosphate buffer, supplemented with sodium diethyldithiocarbamate (DIECA), for inoculation: Mix 1.3 mL of 0.2 M NaH2PO4, 8.7 mL of 0.2 M Na2HPO4, and 0.225 g of DIECA. Make up the volume to 100 mL using ddH20. Adjust pH to 7.6 by adding 1 M NaOH or 1 M HCl solution.</li>
		<li>Harvest 6-8 weeks old PVYN-Wi infected potato cv. Pentland plants from tissue culture in extraction bags with filter net (0.5 g per 6 plants) and add phosphate buffer supplemented with DIECA (4x mass of plant material, mass: volume ratio). Use a hand homogenizer for 1-2 min to obtain a homogeneous solution.</li>
		<li>Lightly dust the first three bottom fully developed leaves of 3-4 weeks old potato plants (from step 1.6) with carborundum powder. 
		NOTE: Adjust the amount of carborundum powder. Too much may cause damage, while too little might result in ineffective infection. Optimally, 0.1 mg/cm of carborundum powder is used, which is approximately 1.5 mg of powder per leaf of average size (approximately 15 cm2).</li>
		<li>Gently rub the leaves with the inoculum (~100 &#181;L per leaf). After 10 min, thoroughly wash the leaves with tap water.
		<ol>
			<li>Be careful not to harm the leaves. Do not exceed the incubation time. Adjust the amount of the inoculum according to leaf size - 6.5 &#181;L/cm, which is approximately 100 &#181;L per leaf of average size (approximately 15 cm2).</li>
		</ol>
		</li>
		<li>Transfer the plants to a growth chamber and grow them under controlled environmental conditions (28 &#176;C in the light and 28 &#176;C in the dark at a relative humidity of 55% &#177; 5%, with 250 &#181;mol/m2/s&#160;radiation and a 16 h photoperiod) for 3 days.</li>
	</ol>
	</li>
</ol>
3. Plant preparation and using the digital microscope for recording lesion development
<ol>
	<li>Transfer the inoculated plants from the growth chamber maintained at 28 &#176;C to a growth chamber at 22 &#176;C 3 days after inoculation, with other conditions as described in step 1.5.</li>
	<li>Select a plant for observation. Immobilize the second inoculated leaf using tape (Figure 1D). Make sure that the desired leaf is fully immobilized before starting with imaging (Figure 1D).</li>
	<li>Install a software application for image capture on the laptop computer. Connect the digital microscope to the computer. Open the software. 
	NOTE: Make sure that the plant, digital microscope, and laptop are positioned on the shelf near the socket to plug in the computer. The decision on the selection of leaves to be monitored might depend on the biological question studied, e.g., the type of process that leads to the formation of limited programmed cell death.</li>
	<li>Adjust the microscope above the immobilized leaf. Focus using the dial on the digital microscope (shown in Figure 1E).
	<ol>
		<li>Ensure that the line of sight is as wide as possible to increase the chances of capturing the formation of the lesion but still magnified enough to spot the lesion&#39;s appearance (typically, 25x magnification is used).</li>
	</ol>
	</li>
	<li>Set the camera settings. Click on the Settings button in the top right corner of the image (Figure 2A, circled part 1) and adjust camera settings: Brightness (60-70), Contrast (10-15), Hue (0), White Balance, Saturation (45-50), Sharpness (0), Gamma (5) (Figure 2B). The settings used in this study were as follows: Brightness (64), Contrast (14), Hue (0), Saturation (47), Sharpness (0), Gamma (5). 
	NOTE: Camera settings should be adjusted to outside light to ensure the best image quality. Settings could be adapted according to the user&#39;s conditions in the growth chamber.</li>
	<li>Set the image capture settings by clicking on the icon for Image Capture (Figure 2C, circled part 2). Click on the button Time-Lapsed Video and set Image Capture every 15 min for 24 h (Figure 2C). 
	NOTE: The interval between the images taken and imaging duration are fully flexible and could be adapted according to the needs of the experiment. In the time between image capture, the LED light is switched off. The LED light is automatically switched on during image capture.</li>
	<li>Start image capture by clicking on the START button (Figure 2C). 
	NOTE: After being transferred from 28 &#176;C to 22 &#176;C, plants should start to develop lesions within 24 h. If not, this could be a sign of ineffective inoculation. Adjust the amount of carborundum powder, ensure that the time of inoculum incubation on leaves was correct, and determine virus abundance in the inoculum by qPCR.</li>
	<li>To save images, select all images and click the Save icon (Figure 2B, circled part 3). Set Export Options. Set DPI at maximum (300) (Figure 2D). After saving, delete all pictures in the program.</li>
	<li>For image analysis, any program for image viewing/editing is sufficient. The usage of free software for image editing, ImageJ, is explained below.
	<ol>
		<li>Import the time sequence of images from a single field of vision (on the top left corner, click File, select Import, and then Image Sequence). Paste the path of the directory of saved pictures and press the OK button to start the conversion.</li>
		<li>After the conversion, the software automatically opens an internal video player showing the final video. Export the video file by clicking on File &#62; Save as option and selecting AVI Format. A small window opens. Set the Frame Rate as 0.3 fps and press OK to save the video as an AVI video file. 
		NOTE: The time of lesion development can also be determined by manually checking all of the images and finding an image where the lesion appears.</li>
	</ol>
	</li>
</ol>

Quantifying Cell Death Initiation Through Continuous Imaging

<ol>
	<li>Karasev, A. V., Gray, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+and+emerging+challenges+of+potato+virus+y+in+potato.">Continuous and emerging challenges of potato virus y in potato.</a> Annual Review of Phytopathology. 51, 571-586 (2013).</li><li>Quenouille, J., Vassilakos, N., Moury, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+virus+Y:+A+major+crop+pathogen+that+has+provided+major+insights+into+the+evolution+of+viral+pathogenicity.">Potato virus Y: A major crop pathogen that has provided major insights into the evolution of viral pathogenicity.</a> Molecular Plant Pathology. 14 (5), 439-452 (2013).</li><li>Szajko, K., 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=The+novel+gene+Ny-1+on+potato+chromosome+IX+confers+hypersensitive+resistance+to+Potato+virus+Y+and+is+an+alternative+to+Ry+genes+in+potato+breeding+for+PVY+resistance.">The novel gene Ny-1 on potato chromosome IX confers hypersensitive resistance to Potato virus Y and is an alternative to Ry genes in potato breeding for PVY resistance.</a> Theoretical and Applied Genetics. 116 (2), 297-303 (2008).</li><li>Szajko, K., Strzelczyk-&#379;yta, D., Marczewski, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ny-1+and+Ny-2+genes+conferring+hypersensitive+response+to+potato+virus+Y+(PVY)+in+cultivated+potatoes:+Mapping+and+marker-assisted+selection+validation+for+PVY+resistance+in+potato+breeding.">Ny-1 and Ny-2 genes conferring hypersensitive response to potato virus Y (PVY) in cultivated potatoes: Mapping and marker-assisted selection validation for PVY resistance in potato breeding.</a> Molecular Breeding. 34 (1), 267-271 (2014).</li><li>Lukan, T., 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=Cell+death+is+not+sufficient+for+the+restriction+of+potato+virus+Y+spread+in+hypersensitive+response-conferred+resistance+in+potato.">Cell death is not sufficient for the restriction of potato virus Y spread in hypersensitive response-conferred resistance in potato.</a> Frontiers in Plant Science. 9, 168 (2018).</li><li>Baebler, &. #. 3. 5. 2. ;., 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+is+an+indispensable+component+of+the+Ny-1+resistance-gene-mediated+response+against+Potato+virus+y+infection+in+potato.">Salicylic acid is an indispensable component of the Ny-1 resistance-gene-mediated response against Potato virus y infection in potato.</a> Journal of Experimental Botany. 65 (4), 1095-1109 (2014).</li><li>Lukan, T., Coll, A., Baebler, &. #. 3. 5. 2. ;., Gruden, K. <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+virus+spread+around+the+cell+death+zone+at+spatiotemporal+resolution+using+confocal+microscopy.">Analysis of virus spread around the cell death zone at spatiotemporal resolution using confocal microscopy.</a> Methods in Molecular Biology. 2447, 261-270 (2022).</li><li>Vinterhalter, D., Dragi&#252;evi&#252;, I., Vinterhalter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potato+in+vitro+culture+techniques+and+biotechnology.">Potato in vitro culture techniques and biotechnology.</a> Fruit, Vegetable and Cereal Science and Biotechnology. 2, 16-45 (2008).</li><li>Wang, W., 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=Timing+of+plant+immune+responses+by+a+central+circadian+regulator).">Timing of plant immune responses by a central circadian regulator).</a> Nature. 470, 110-115 (2011).</li><li>Roden, L. C., Ingle, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lights,+rhythms,+infection:+The+role+of+light+and+the+circadian+clock+in+determining+the+outcome+of+plant-pathogen+interactions.">Lights, rhythms, infection: The role of light and the circadian clock in determining the outcome of plant-pathogen interactions.</a> Plant Cell. 21 (9), 2546-2552 (2009).</li><li>Srivastava, D., 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=Role+of+circadian+rhythm+in+plant+system:+An+update+from+development+to+stress+response.">Role of circadian rhythm in plant system: An update from development to stress response.</a> Environmental and Experimental Botany. 162, 256-271 (2019).</li><li>Mulaosmanovic, E., 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=High-throughput+method+for+detection+and+quantification+of+lesions+on+leaf+scale+based+on+trypan+blue+staining+and+digital+image+analysis.">High-throughput method for detection and quantification of lesions on leaf scale based on trypan blue staining and digital image analysis.</a> Plant Methods. 16, 62 (2020).</li><li>Martinelli, F., 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=Advanced+methods+of+plant+disease+detection.+A+review.">Advanced methods of plant disease detection. A review.</a> Agronomy for Sustainable Development. 35, 1-25 (2015).</li><li>Ali, M., Bachik, N., Muhadi, N. A., Tuan Yusof, T. N., Gomes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-destructive+techniques+of+detecting+plant+diseases:+A+review.">Non-destructive techniques of detecting plant diseases: A review.</a> Physiological and Molecular Plant Pathology. 108, 101426 (2019).</li><li>Sankaran, S., Mishra, A., Ehsani, R., Davis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+advanced+techniques+for+detecting+plant+diseases.">A review of advanced techniques for detecting plant diseases.</a> Computers and Electronics in Agriculture. 72 (1), 1-13 (2010).</li><li>Rowarth, N. M., 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=RNA-Seq+analysis+reveals+potential+regulators+of+programmed+cell+death+and+leaf+remodelling+in+lace+plant+(Aponogeton+madagascariensis).">RNA-Seq analysis reveals potential regulators of programmed cell death and leaf remodelling in lace plant (Aponogeton madagascariensis).</a> BMC Plant Biology. 21 (1), 375 (2021).</li></ol>

The authors declare that they have no conflicts of interest.

The demonstrated protocol allows the user to accurately determine the cell death initiation rate by continuously imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope. Even though there are numerous ways of monitoring lesion and plant disease occurrence12,13,14,15, this protocol presents the advantage of light-independent measuring without disturbing the plant&#39;s circadian rhythm, as the light is switched off between measurements.
After the inoculation, plants should grow at 28 &#176;C for 3 days. Ny-1 resistance gene, which induces a hypersensitive response, is temperature-dependent, and in plants grown at higher temperatures, leads to abortion of resistance, which is manifested as lack of lesion formation and systemic virus spread3. After plants are transferred to 22 &#176;C, cell death is initiated, so for accurate results, observation with a digital microscope should begin as soon as possible after this transfer. Another crucial step in the preparation of the plant for imaging is the immobilization of the leaf (Figure 1D), as the plant will continue to grow during imaging, which could move the observed leaf out of focus, or such set-up will not give desired results.
If the described protocol is used on transgenic plants with altered components of interest, hypothesized to be involved in cell death initiation, the protocol enables the user to determine if the decreased level of a studied component affects the rate of cell death initiation. By that, components involved in cell death initiation can be identified in pathosystems, where programmed cell death occurs, using this protocol. Other methods for identifying these components are, for example, transcriptomic analysis such as RNA-seq&#160;or various forms of microscopy, which can be expensive and time-consuming16. The method described in this protocol allows for easy and inexpensive identification of components involved in cell death initiation by observing differences in cell death initiation rates between transgenic and control plants. Optimally, in such a set-up, two digital cameras must be used, as a transgenic plant should be analyzed in parallel with a control plant within the same experiment.
In this protocol, PVY strain N-Wilga was used; however, other strains of this virus, for example, GFP-tagged PVY (PVY-N605(123)-GFP)7, could also be used. Moreover, other pathosystems, which result in programmed cell death development could be studied using this protocol with minor modification.

Potato is one of the world&#39;s most widely grown food crops, fourth place behind rice, wheat, and corn. However, potato production can be severely affected by potato virus Y (PVY), which is currently considered its most important virus pathogen1,2. In potato plants cv. Rywal, several strains of PVY (including PVY strain N-Wilga) trigger hypersensitive response (HR)-conferred resistance, where the pathogen&#39;s restriction to the infection site manifests as necrotic lesions on inoculated leaves3. In this pathosystem, HR is mediated by Ny-1 resistance gene, which is temperature-dependent, as plants grown at lower temperatures efficiently develop necrotic lesions, whereas in plants grown constitutively at elevated (28 &#176;C) temperature, abortion of resistance is demonstrated as lack of lesion formation and systemic virus spread3,4. When the plants are transferred to a lower temperature (22 &#176;C), cell death is initiated, which can be exploited to follow the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance.
This protocol demonstrates a simple method for the determination of cell death initiation rate using a digital microscope. By imaging the inoculated leaves after transferring the plant from 28 &#176;C to 22 &#176;C, a digital microscope enables continuous observation of the leaf in desired intervals. Unlike the use of other methods (for example, confocal microscopy or observation of lesion formation with the naked eye), this allows determination of the exact time of lesion formation and therefore, the cell death initiation rate up to minutes exactly, as opposed to hours in aforementioned methods5,6. Digital microscope use is also independent of light and can therefore be used during day and night. This protocol can also be used to identify components involved in cell death initiation or to determine the effects of different components on cell death initiation rate if used plants are transgenic and have altered levels of components of interest.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcohol burner</td>
			<td>Mikro+Polo</td>
			<td>SH-234002455</td>
			<td>For tweezers and scalpel sterilization</td>
		</tr>
		<tr>
			<td>Autoclave A-21 CAV</td>
			<td>Kambi<img alt="Equation 1" src="/files/ftp_upload/65824/65824eq01.jpg" style="margin: auto;" /></td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>Becton, Dickinson and Company</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Carborundum powder</td>
			<td>VWR Chemicals</td>
			<td>22505297</td>
			<td></td>
		</tr>
		<tr>
			<td>DinoCapture 2.0&#160;</td>
			<td>Dino-Lite</td>
			<td>Version 2.0</td>
			<td>software for digital microscope</td>
		</tr>
		<tr>
			<td>Dino-Lite Edge AM7915MZTL digital microscope</td>
			<td>AnMo Electronics Corporation</td>
			<td>AM7915MZTL</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 70%</td>
			<td>Stella Tech</td>
			<td>P94000</td>
			<td>For tweezers and scalpel sterilization</td>
		</tr>
		<tr>
			<td>Extraction bags</td>
			<td>Bioreba</td>
			<td>420100</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth chamber FS-WI</td>
			<td>Photon Systems Insturments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand homogenizer</td>
			<td>Bioreba</td>
			<td>400010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hawita Special Substrate</td>
			<td>HAWITA Gruppe</td>
			<td>2000000071701</td>
			<td>Ready to use substrate, made using peat (H4-H6 and H6-H8)</td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>Merck</td>
			<td>109057</td>
			<td></td>
		</tr>
		<tr>
			<td>Label tape</td>
			<td>Sigma</td>
			<td>L8144-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Laptop computer with installed DinoCapture 2.0&#160;</td>
			<td>HP</td>
			<td>Z2V77EA#BED</td>
			<td>Computer needs to be transferable as experiment takes part in a growth chamber</td>
		</tr>
		<tr>
			<td>Murashige and Skoog medium</td>
			<td>Duchefa Biochemie</td>
			<td>M02220100</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Emsure</td>
			<td>1065860500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Emsure</td>
			<td>1064700250</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette&#160; 0.5 mL</td>
			<td>Brand</td>
			<td>21500209</td>
			<td></td>
		</tr>
		<tr>
			<td>pH-meter</td>
			<td>Mettler Toledo</td>
			<td>ML1601</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic boxes&#160;</td>
			<td>Cvetlice Dornig</td>
			<td>VCG10.5</td>
			<td>Radius = 10.5 cm</td>
		</tr>
		<tr>
			<td>Plastic pots&#160;</td>
			<td>Lab Associates</td>
			<td>DIS40003</td>
			<td>Radius = 11.5 cm (top), Radius = 9.8 cm (bottom)</td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>Kemika d.d.</td>
			<td>1800408</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Diethyldithiocarbamate (DIECA)</td>
			<td>Sigma-Aldeich</td>
			<td>228680</td>
			<td>Sodium diethyldithiocarbamate trihydrate, ACS reagent</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Merck</td>
			<td>106462</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical blades</td>
			<td>Braun</td>
			<td>4511733633</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Braun</td>
			<td>BD033R</td>
			<td></td>
		</tr>
	</tbody>
</table>

studying cell death initiation using a digital microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

This study demonstrates a step-by-step protocol for studying cell death initiation through lesion occurrence on potato cv. Rywal, with a digital microscope. This enables determining the exact time of programmed cell death initiation.
Plants that have developed roots were put in soil 2 weeks after potato cv. Rywal micropropagation (Figure 1A,B). After 3-4 weeks of growth under described conditions, plants with at least 3-4 fully developed leaves with visible leaflets that looked healthy, with no signs of abscission, were used for further analysis (Figure 1C). Using a digital microscope as described in this protocol, we observed the same area on the inoculated leaf at 15-min-intervals and determined the lesion occurrence and expansion in time (Figure 3). The lesion occurred at 15 h 30 min (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65824/65824fig01.jpg" /> 
Figure 1: Plant preparation for analysis with a digital microscope. (A) A plastic box with MS 30 medium and potato cv. Rywal plant explants containing nodes. (B) Potato cv. Rywal plant in soil (2 weeks after micropropagation). (C) Potato cv. Rywal plant, ready for inoculation (4 weeks after being put in soil), having at least three fully developed leaves. (D) Second inoculated leaf (arrow) of potato cv. Rywal plant positioned and immobilized (arrow) with tape. (E) Plant positioned under the digital microscope with the arrow pointing to the dial used for focusing. <a href="https://www.jove.com/files/ftp_upload/65824/65824fig01large.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/65824/65824fig02.jpg" /> 
Figure 2: Digital software setting for recording lesion development. (A) Software interface - circled with red are options for the button for (1) camera&#160;settings, (2) image&#160;capture&#160;settings, and (3) saving&#160;images. (B) Window with camera settings, which opens with a click on (1) in panel A. Brightness, Contrast, Saturation, Sharpness, and Gamma should be properly adjusted. (C) Window with image capture settings, which opens with a click on (2) denoted in panel A. (D) Window with image saving settings, which opens with a click on (3) denoted in panel A. <a href="https://www.jove.com/files/ftp_upload/65824/65824fig02large.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/65824/65824fig03.jpg" /> 
Figure 3: Lesion formation on the inoculated leaf observed under the digital microscope. Images of the central part of PVY-inoculated potato leaf at 23.6x magnification as seen under the digital microscope, taken at intervals of 5 min. Inoculated plants were put at 28 &#176;C for 3 days, and on the third day, the observation with a digital microscope at 22 &#176;C started at 7:00. (A) At 21:02, the lesion is not yet visible, (B) 90 min later, at 22:32, the lesion is visible. (C) The lesion expansion was observed at 01:02 and (D) 07:32 the next morning. Experiment was repeated two times, and the lesions occurred 8 h 15 min, and 12 h after cell death initiation, respectively. <a href="https://www.jove.com/files/ftp_upload/65824/65824fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response at JoVE.com

Author Spotlight: A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...

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1.2K Views.  National Institute of Biology. Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction. 

Video: Author Spotlight: A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response 

Studying Aggression in Drosophila (fruit flies)

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources. This complex social behavior is influenced by genetic, hormonal and environmental factors. In many organisms, aggression is critical to survival but controlling and suppressing aggression in distinct contexts also has become increasingly important. In recent years, invertebrates have become increasingly useful as model systems for investigating the genetic and systems biological basis of complex social behavior. This is in part due to the diverse repertoire of behaviors exhibited by these organisms. In the accompanying video, we outline a method for analyzing aggression in Drosophila whose design encompasses important eco-ethological constraints. Details include steps for: making a fighting chamber; isolating and painting flies; adding flies to the fight chamber; and video taping fights. This approach is currently being used to identify candidate genes important in aggression and in elaborating the neuronal circuitry that underlies the output of aggression and other social behaviors.

Studying Aggression in Drosophila fruit flies

Aggression is an innate behavior that evolved in the framework of defending or obtaining resources.  This complex social behavior is influenced by ...

Cell Capture Using a Microfluidic Device

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

Application of Ultrasound and Shear Wave Elastography Imaging in a Rat Model of NAFLD/NASH

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat accumulation (steatosis) and inflammation leading to fibrosis. Preclinical models closely recapitulating human NASH/NAFLD are essential in drug development. While liver biopsy is currently the gold standard for measuring NAFLD/NASH progression and diagnosis in the clinic, in the preclinical space, either collection of whole liver samples at multiple timepoints during a study or biopsy of liver is needed for histological analysis to assess the disease stage.
Conducting a liver biopsy mid-study is an invasive and labor-intensive procedure, and collecting liver samples to assess disease level increases the number of research animals needed for a study. Thus, there is a need for a reliable, translatable, non-invasive imaging biomarker to detect NASH/NAFLD in these preclinical models. Non-invasive ultrasound-based B-mode images and Shear Wave Elastography (SWE) can be used to measure steatosis as well as liver fibrosis. To assess the utility of SWE in preclinical rodent models of NASH, animals were placed on a pro-NASH diet&#160;and underwent non-invasive ultrasound B-mode and shear wave elastography imaging to measure hepatorenal (HR) index and liver elasticity, measuring progression of both liver fat accumulation and tissue stiffness, respectively, at multiple time points over the course of a given NAFLD/NASH study.
The HR index and elasticity numbers were compared to histological markers of steatosis and fibrosis. The results showed strong correlation between the HR index and percentage of Oil Red O (ORO) staining, as well as between elasticity and Picro-Sirius Red (PSR) staining of livers. The strong correlation between classic ex vivo methods and in vivo imaging results provides evidence that shear wave elastography/ultrasound-based imaging can be used to assess disease phenotype and progression in a preclinical model of NAFLD/NASH.

This protocol describes the use of an enhanced ultrasound technique to non-invasively observe and quantify liver tissue changes in rodent models of nonalcoholic fatty liver disease.

Nonalcoholic Steatohepatitis (NASH) is a condition within the spectrum of Non-Alcoholic Fatty Liver Disease (NAFLD), which is characterized by liver fat ...