The overall goal of this procedure is to observe the movement of fluorescently tagged proteins on plant cell surfaces with variable angle epi fluorescence microscopy. This vessel can help us progress in plant cell biology field, such as how do protein work on plate cell surfaces. The main vantage of this technique is that it allows us to visualize real time dynamics of fluorescent attack proteins Before beginning the procedure.
Calibrate the laser centering and focusing of the microscope according to the manufacturer's instructions. Then use an objective lens free light path to locate the center of the microscopy room ceiling and mark the position with a colored seal. Next, illuminate the ceiling with an objective lens and move the illuminated region to the center position.
Then focus the laser and fine tune the position of the laser to the center of the ceiling. The centering and focusing of the laser is very important for successful variable angle epi scent microscope or VAEM observation. Users should be trained by a professional staff person from the microscope company before starting an experiment.
When the microscope is ready, dispense 30 microliters of basal buffer onto the center of a 76 by 26 millimeter glass slide. Then use dissecting scissors to remove a co leadin from a seven day old seedling and float the co leadin with the observation side up on the basal buffer drop. Next, add 30 microliters of basal buffer onto the center of an 18 by 18 millimeter.
Cover glass and turn the cover glass upside down. Gently surface tension will prevent the drop from falling, holding the cover glass with tweezers on one edge. Place the opposite edge onto the glass slide so that the buffer is over the co lead-in.
Then keeping the edge of the cover glass on the slide. Adjust the position of the drop so that it is directly above the olein sample and drop the cover glass. If there are no air bubbles, use lint-free tissues to wipe off the excess buffer and immediately transfer the preparation to the microscope.
Using brightfield illumination, select the cells for observation. Next, confirm that the fluorescent protein can be observed and use epi fluorescence illumination to set the Z axis position at the cell surface. To perform VAEM, use the controller box to incline the entry angle of the laser beam.
Gradually while monitoring the live image carefully. Initially, the image will appear blurred. As the laser angle increases, the VAEM image will become less blurry, eventually producing a clear image.
At this point, stop increasing the laser angle. Now fine. Tune the laser entry angle to obtain a better image, adjusting the optical and image sensor parameters as necessary.
Then using the commercial microscope software, acquire a movie as a multi-page TIFF image file. Here the movie shows GFP labeled dots blinking on the surface of STA model cells. To quantify the GFP labeled DOT residence time using Fiji, go to the file open menu, and open the acquired multi-page tip file.
Use the straight line selection tool to place a line on the side of interest. Next, use the image stacks dynamic res slice plugin to generate a graph image. As the line changes, the graph image will change dynamically.
Save the image as a TIFF file under the file, save as tiff menu. Then to perform a noise reduction, go to the process filters Gaussian blur menu, and apply a Gaussian filter to the chronograph images. The sigma radius parameter should be tuned as necessary.
Using the image adjust threshold tool, segment the signal regions by thresholding and open the analyze set measurements menu. Next, check the bounding rectangle in the set measurements window, selecting the minimum number of pixels possible. Then under the analyze, analyze particles menu.
Measure the time of the dot of interest against the Y axis and check the display results and add to manager boxes to view the data values. Finally, under the results table menu, select file, save as and process the dataset of dot image durations using the appropriate analysis software. Here, the residence times of 185 GFP patrol one dots from 12 independent subsidiary cells in eighth edin as measured by chm graph analysis are shown as illustrated in the graph.
The fitted density function revealed that most of the GFP patrol one dots were resident in a subsidiary cell for between two and 10 seconds with a peak of around 3.5 seconds. This suggests a fast exocytosis endocytosis of the proton pumps on the subsidiary cell surface. After working this video, you should have a good understanding of how to visualize and quantify ary type protein dynamics on plant cell surface using variable angle epi fluorescence microscopy.
