Name: real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy
Uploaded: 2015-12-12T14:00:00.000+00:00
Duration: 6 min 31 s
Description: Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

I am grateful to Dr. Masaru Fujimoto for his technical suggestions for VAEM. I am also grateful to Prof. Koh Iba and Dr. Mimi Hashimoto-Sugimoto for providing GFP-PATROL1 transgenic plants, and discussions about PATROL1. I thank Prof. Seiichiro Hasezawa for his continuing support of my work. This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 25711017.

1. Preparation of Seedlings
<ol>
	<li>Sterilize the seeds.
	<ol>
		<li>Prepare the sterilization solution by adding 500 &#181;l NaClO (available chlorine: 5.0%) and 1 &#181;l 10% Triton X-100 to 500 &#181;l sterile water.</li>
		<li>Place ca. 10 transgenic A. thaliana seeds carrying GFP-PATROL18 into a 1.5-ml tube.</li>
		<li>Add 1 ml 70% ethanol solution, and mix well by inverting five times. Leave for 1 min.</li>
		<li>Observe the seeds sink to the bottom of the tube. In a clean laminar flow cabinet, gently remove the 70% ethanol using a micropipette, and add 1 ml sterilization solution. Mix well by inverting five times and leave for 5 min.&#160;</li>
		<li>Wash the seeds. Still working under aseptic conditions on a clean bench, gently remove the solution using a micropipette, and add 1 ml sterile water. Repeat this five times. The sterilized seeds may be stored in sterile water at 4 &#176;C for 2 days.</li>
	</ol>
	</li>
	<li>Sow the sterilized seeds on a 0.5% gellan gum-solidified 1/2 Murashige and Skoog medium plate (pH 5.8)9. Tape the lid onto the plate using two layers of surgical tape.</li>
	<li>Incubate the plate in the dark at 4 &#176;C with a cold storage chamber, O/N.</li>
	<li>Transfer the plate into a growth chamber set at 23.5 &#176;C with a 12-hr/12-hr light-dark cycle using 100 &#181;mol m&#8722;2 sec&#8722;1 white lights, and incubate for 7 days. Seedlings with cotyledons of around 1 mm long can then be harvested.</li>
</ol>
2. Sky Drop Mounting of Cotyledon Specimens
NOTE: An important factor in specimen preparation for VAEM observation is avoiding the inclusion of air bubbles between the specimen and the cover glass. Bubbles greatly reduce the image quality of VAEM by causing differences in the refractive index. A simple method, which we have called &#8216;sky drop&#8217; mounting, can be used to avoid bubbles between the A. thaliana cotyledons and the cover glass. This should be done immediately prior to observation.
<ol>
	<li>Place 30 &#181;l of basal buffer [5 mM 2-(N-morpholino)-ethanesulfonic acid-Tris (MES-Tris) at pH 6.5, 50 mM KCl, 100 &#181;M CaCl2] on the center of a glass slide (size: 76&#215;26 mm, thickness: 1.0&#8211;1.2 mm).</li>
	<li>Remove a cotyledon from a 7-day-old seedling using dissecting scissors. Float the cotyledon with the observation side facing up on the basal buffer drop (Figure 1, step 1).</li>
	<li>Place 30 &#181;l of basal buffer on the center of a cover glass (size: 18&#215;18 mm, thickness: 0.12&#8211;0.17 mm) (Figure 1, step 2). Turn the cover glass upside down gently. Surface tension will prevent the buffer drop from falling off. Hold the cover glass with tweezers on one edge. Place the opposite edge on the glass slide so that the buffer drop is approximately above the cotyledon.</li>
	<li>With the edge of the cover glass still on the slide, and still holding the opposite edge with tweezers, adjust the position of the buffer drop under the cover glass so that it is directly above the cotyledon sample (Figure 1, step 3). Let go of the cover glass. Mount a drop on the sample resulting in a preparation without air bubbles.</li>
	<li>Wipe off excess buffer using lint-free tissues. Observe the preparation immediately (see step 3). 
	NOTE: There is no need to seal the samples.</li>
</ol>
3. VAEM Observation and Movie Acquisition
NOTE : The TIRF microscope system9 used in the present study is described as follows: An inverted microscope is equipped with a TIRF unit and a TIRF objective lens with a numerical aperture of 1.49. For computerized control of the laser entry angle, a control box is used. Green fluorescent protein (GFP) is excited with a 488 nm optically pumped semiconductor laser, and the fluorescence is detected through a 510&#8211;550 nm band-pass filter to prevent autofluorescence from chloroplasts. The measured maximum value of fiber output power is 13.0&#8211;13.5 mW. For detection, an electron multiplying charge-coupled device (EM-CCD) camera head system and a C-mount camera magnification change unit are used.
<ol>
	<li>Calibrate the laser centering and focusing according to the manufacturer&#8217;s instructions. 
	NOTE: This step is crucial for precise VAEM observations. It is strongly recommended that periodic checks of the laser path adjustment procedures, appropriate to your microscope, are carried out.
	<ol>
		<li>Determine the central position illuminated with an objective lens-free light path on the ceiling of the microscopy room. To mark the central position, put a colored circle seal on the ceiling (Figure 2, step 1, 2).</li>
		<li>Illuminate the ceiling with an objective lens (Figure 2, step 3, 4). Move the illuminated region to the center position (Figure 2, step 5). Focus the laser (Figure 2, step 6). Fine-tune the position of the focused laser to the center (Figure 2, step 7).</li>
	</ol>
	</li>
	<li>Set a specimen on the stage of the microscope and select cells for observation using bright field illumination.</li>
	<li>Check that the fluorescent protein can be observed in the cells, and set the z-axis position at the cell surface using epifluorescence illumination (Figure 3A).</li>
	<li>Perform VAEM observations.
	<ol>
		<li>Incline the entry angle of the laser beam gradually with the controller box. At the same time, monitor the live image carefully. Initially, the image will be blurred (Figure 3B).</li>
		<li>As the laser angle increases, the VAEM image will become less blurred, eventually producing a clear image (Figure 3C and D). At this point, stop increasing the laser angle. If the fluorescence signal is lost, decrease to a shallower angle.</li>
		<li>Fine-tune the laser entry angle to obtain a better image. Fine-tuning of the z-axis position may also improve the image. If necessary, adjust the optical parameters (laser power, wavelength and filter set) and image sensor parameters [image size, exposure time, gain, digitizer and electron-multiplying (EM) gain]. 
		NOTE: In the case of the TIRF microscope equipment described above, representative parameters are as follows. Magnification of lens just in front of the camera: 2X; Laser output: 1.0 mW; Image size: 512 &#215; 512 pixels; Exposure time: 100 msec; Gain: 5&#215;; Digitizer: 11 MHz; and EM gain: 100.</li>
	</ol>
	</li>
	<li>&#160;Acquire a movie as a multi-page TIFF image file using the commercial microscope software. Here, the movie is of GFP-labeled dots blinking on the surface of stomatal cells. 
	NOTE: Representative image acquisition conditions are as follows. Acquisition Mode: Stream to RAM; Number of frames: 600; and multi-page TIFF file size: ~302 MB.</li>
</ol>
4. Kymograph Analysis for Quantification of GFP-labeled Dot Residence Time Using Fiji Software
<ol>
	<li>Install Fiji (&#8216;Fiji is Just ImageJ&#8217;) software10 using the authors&#8217; instructions (http://fiji.sc/Fiji).</li>
	<li>Run Fiji and open the acquired multi-page TIFF file using the Fiji menu &#8220;File-Open&#8221;.</li>
	<li>Place a line on the site of interest, using the Fiji tool bar menu &#8220;Straight Line Selection Tool&#8221;. Optionally, use the &#8220;Segmented Line Selection Tool&#8221; or &#8220;Freehand Line Selection Tool&#8221;. In the results presented here, a straight line of 100 pixels (corresponding to 8.0 &#181;m) was placed on a subsidiary cell (Figure 4A).</li>
	<li>Make a kymograph image using the Fiji menu &#8220;Image-Stacks-Dynamic Reslice&#8221; (http://fiji.sc/Dynamic_Reslice) (Figure 4B). Optionally, &#34;Flip Vertically&#8221; and &#8220;Rotate 90 Degrees&#8221; in a check box window are also available (not used in the results presented here). The x- and y-axis in the kymograph image indicate the line position (100 pixels corresponding to 8.0 &#181;m) and observation time (600 pixels corresponding to 60 sec), respectively. If the kymograph image is not satisfactory, the position and type (straight, segmented and freehand) of the line on the multi-page image may be changed. As the line changes, the kymograph image dynamically changes. Save a kymograph image as a TIFF file using the Fiji menu &#8220;File-Save As-Tiff&#8221;.</li>
	<li>Measure the length of the blob in the orientation of time axis.
	<ol>
		<li>To perform noise reduction, apply a Gaussian filter to the kymograph images using the Fiji menu &#8220;Process-Filters-Gaussian Blur&#8221;. The &#8220;Sigma (Radius)&#8221; parameter is tunable (1 pixel radius is used for the presented results).</li>
		<li>Segment the signal regions by thresholding, using the Fiji menu &#8220;Image-Adjust-Threshold&#8221;. 
		NOTE: There are several thresholding algorithms to choose from. In presented results, the &#8220;Yen&#8221; algorithm was chosen (Figure 4C).</li>
		<li>Prepare to measure the time (y)-axis length of the blob using the menu &#8220;Analyze-Set Measurements&#8221;. Check the &#8220;Bounding rectangle&#8221; in the &#8220;Set Measurements&#8221; window. Ideally the area set should be as small as possible, to exclude noise. Here, the minimum area was set at 50 pixels. Measure the time (y)-axis length of the blob using the menu &#8220;Analyze-Analyze Particles&#8221;. To obtain the result values and prove credibility of the measurement regions, check the &#8220;Display results&#8221; and &#8220;Add to Manager&#8221; boxes.</li>
		<li>Values in the &#8220;Height&#8221; column are the time (y)-axis lengths for the measured blob (Figure 4D). Save the values using the Results table menu &#8220;File-Save As&#8221; and calculate and process the dataset of blob image durations using a statistical package and/or spreadsheet software (Figure 4E).</li>
	</ol>
	</li>
</ol>

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The author has nothing to disclose.

In this video article, protocols are given for monitoring and measuring the dynamic behavior of GFP-PATROL1 dots on the stomatal complex of Arabidopsis thaliana. As shown here, VAEM observation is a powerful tool for live imaging of plant cell surfaces. Under the experimental conditions used here for GFP-PATROL1 monitoring, there was very little fluorescence photobleaching in the sample used for 1 min of video capture, because the highly sensitive EM-CCD permits the use of a relatively weak excitation laser in V...

The plant cell surface, including the plasma membrane and its immediately adjacent cytoplasm, is the main region of a plant cell&#8217;s perception and integration of biotic and abiotic cues from the extracellular environment. In response to these cues, cell surface components including plasma membrane proteins and the cortical cytoskeleton undergo dynamic changes, on a time scale of seconds to minutes1-4. Thus, real-time and high-resolution imaging of fluorescent proteins on the cell surface can illuminate a plant&#8217;s responses to environmental cues at the molecular level.
Confocal laser scanning microscopy is a powerful tool for determination of fluorescently-tagged protein localization3, however, it is often difficult to monitor the real-time protein dynamics because of its relatively long capturing times. An emerging technique for real-time monitoring of proteins in the plant cell is variable angle epifluorescence microscopy (VAEM), which is an adaptation of equipment usually used for total internal reflection fluorescence (TIRF) microscopy. In TIRF microscopy, the fluorescence-excitation light source is an evanescent light field that is generated when the entry angle of the laser is shallow enough to totally internally reflect light at the glass&#8211;water interface. The penetration depth of the evanescent light field is around 100 nm. TIRF microscopy is an outstanding tool for single molecule imaging, such as the detection of exocytosis in animal cells5. However, evanescent light cannot reach plasma membranes or the cortical cytoplasm in plant cells, because they have thick cell walls. Recently, TIRF microscopy equipment has been adapted by plant cell biologists, observing that a laser, if angled slightly more deeply than when being used to induce total internal reflection phenomena, could excite the surface of plant cell samples, resulting in high-quality plant cell imaging6,7. The excitation illumination depth is varied by adjusting the entry angle of the laser; therefore, this technique is described as VAEM. This optical system is also called variable angle TIRF microscopy (VA-TIRFM) because there is a possibility that total reflection may take place at the cell wall-periplasm interface7, however, the term VAEM is used in this article, as per the first report in plants6.
The goal of this protocol is to demonstrate practical procedures for using VAEM to visualize fluorescently-tagged protein dynamics on plant cell surfaces. Additionally, an image analysis protocol to quantify the residence time (duration of presence) of molecules is described for VAEM movie analysis. GFP-PATROL1 dot blinking on stomatal complex cells in Arabidopsis thaliana cotyledons is used as an example. PATROL1 was identified by forward genetic approaches as a causal gene of a stomatal response defect mutant in A. thaliana8. PATROL1 is a plant homolog of MUNC-13, which is a priming factor in synapse vesicle exocytosis8. In response to environmental cues, such as light or humidity, it is thought that PATROL1 reversibly regulates the delivery of a proton pump to plasma membranes in the stomatal complex. Stomatal complexes each comprise a pair of guard cells8 and subsidiary cells9, and they require a proton pump for stomatal movement. In these cells, GFP-tagged PATROL1 localizes to dot-like structures that remain on the plasma membrane for less than 1 min9.

<table><tbody><tr><td>Inverted microscope</td><td>Olympus</td><td>IX-73</td><td></td></tr><tr><td>TIRF unit</td><td>Olympus</td><td>IX3-RFAEVAW</td><td></td></tr><tr><td>TIRF objective lens&amp;nbsp;</td><td>Olympus</td><td>UAPON 100 &amp;times; OTIRF&amp;nbsp;</td><td>NA = 1.49</td></tr><tr><td>Laser angle control box</td><td>Chuo Seiki</td><td>QT-AK</td><td></td></tr><tr><td>Optically pumped semiconductor laser</td><td>Coherent</td><td>Sapphire&lt;sup&gt;TM&lt;/sup&gt; LP USB 488-20 CDRH Laser</td><td></td></tr><tr><td>510&amp;ndash;550 nm band-pass filter</td><td>Olympus</td><td>U-FBNA</td><td></td></tr><tr><td>EM CCD camera</td><td>Hamamatsu Photonics</td><td>ImagEM C9100-13</td><td></td></tr><tr><td>C-mount camera magnification change unit&amp;nbsp;</td><td>Olympus</td><td>U-TVCAC</td><td></td></tr><tr><td>MetaMorph software</td><td>Molecular Devices</td><td>MetaMorph version 7.7.11.0</td><td></td></tr><tr><td>TIRF microscopy manual</td><td>Olympus</td><td>AX7385</td><td>Instructions: Total Internal Reflection Illumination System (Printed in Japan on August 24, 2012)</td></tr><tr><td>Immersion oil</td><td>Olympus</td><td>Immersion Oil Typr-F</td><td>ne = 1.518 (23 degrees)</td></tr></tbody></table>

real-time imaging of plant cell surface dynamics with variable-angle epifluorescence microscopy

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and cytoskeletal rearrangement. Real-time and high-resolution image analysis of such intracellular events will increase the understanding of plant cell biology at the molecular level. Variable angle epifluorescence microscopy (VAEM) is an emerging technique that provides high-quality, time-lapse images of fluorescently-labeled proteins on the plant cell surface. In this article, practical procedures are described for VAEM specimen preparation, adjustment of the VAEM optical system, movie capturing and image analysis. As an example of VAEM observation, representative results are presented on the dynamics of PATROL1. This is a protein essential for stomatal movement, thought to be involved in proton pump delivery to plasma membranes in the stomatal complex of Arabidopsis thaliana. VAEM real-time observation of guard cells and subsidiary cells in A. thaliana cotyledons showed that fluorescently-tagged PATROL1 appeared as dot-like structures on plasma membranes for several seconds and then disappeared. Kymograph analysis of VAEM movie data determined the time distribution of the presence (termed &#8216;residence time&#8217;) of the dot-like structures. The use of VAEM is discussed in the context of this example.

In this video article, the protocols for VAEM observation of GFP-PATROL1 in A. thaliana cotyledon stomatal complex cells are provided. Sky drop mounting is a simple preparation method that may help reduce the occurrence of air bubbles in VAEM preparations of A. thaliana cotyledons (Figure 1). Overtilting of the entry laser and/or z-positioning of specimens for VAEM will provide an unclear image. If that happens, it is recommended to start again from a position immediately above the samp...

Watch this Scientific Journal Video about Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy at JoVE.com

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

The goal of this protocol is to demonstrate how to monitor fluorescently-tagged protein dynamics on plant cell surfaces with variable-angle epifluorescence microscopy, showing blinking dots of GFP-tagged PATROL1, a membrane trafficking protein, in the cell cortex of the stomatal complex in Arabidopsis thaliana.

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

real-time-imaging-plant-cell-surface-dynamics-with-variable-angle

Research

JoVE Journal

Biology

9.0K Views.  The University of Tokyo.  Het doel van dit protocol is om te demonstreren hoe fluorescent-gelabelde eiwit dynamiek op plantencel oppervlakken met een variabele hoek epifluorescentiemicroscopie, waaruit blijkt knipperende stippen van GFP-gelabeld PATROL1, een membraan mensenhandel eiwit te controleren, in de cel cortex van de stomatale complex in Arabidopsis thaliana. 

Video: Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells1. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface2. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching2,3. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups.
At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures4-8. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells.
We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

TIRF microscopy has emerged as  a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules  in vitro and in living cells1. ...

Bioengineering

Live Confocal Imaging of Developing Arabidopsis Flowers

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

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

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

Developmental Biology

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and ...

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In ...

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 ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

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.

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.

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

Spatiotemporal Investigation of ERL1 Dynamics via Confocal Imaging

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Author Spotlight: Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

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