The overall goal of this procedure is to observe the dynamic changes in the protein FTSZ during cell division in bacteria. This is accomplished by applying bacillus subtles cells expressing the FTS said GFP fusion to the surface of a small aros pad. The pad is then inverted and transferred to a Petri dish with a glass cover slip bottom.
The second step is to acquire time-lapse images of the bacteria during cell division using a 3D structured illumination microscopy or 3D SIM imaging system. Next, the quality of the raw data is assessed and images with greater than 30%decrease in intensity due to photobleaching are discarded. The final step is to quantitatively analyze the changes in the protein localization during cell division by measuring the fluorescence intensity distribution of the F-T-S-Z-G-F-P protein over time.
Ultimately fast 3D SIM is used to show how dynamic the dione proteins are during cell division in bacteria. The main advantage of this technique over existing techniques like confocal microscopy, is that we get three dimensional information about the changes that occur in cell division in life cells. Though this method provides insight into bacterial cytokinesis, it could also be applied to other systems where we want to look at dynamic protein changes such as M mammalian cells.
We first had the idea for this when we saw traditional 3D sim images of fixed bacterial cells showed that the FDZ protein was not homogeneously distributed around the zed ring. We wanted to further examine if this distribution was linked to changes that occurred during Zed ring constriction. Generally, individuals who are new to this technique will struggle because of a failure to match the refractive index of their oil to their sample, and this will lead to poor quality images.
To prepare bacillus subtlest cells for imaging, grow the cells until mid exponential phase as detailed in the text protocol. Next, attach an adhesive frame onto a standard glass microscope slide by first removing the thin polyester sheet. Then apply the exposed adhesive surface of the frame to the surface of the microscope slide to effectively create a square well on top of the slide, leave the thick polyester sheet attached to the frame to prevent sticking of the cover slip in subsequent steps.
Next, melt an aro solution in the microwave until boiling pipette 67 microliters of the melted aros into the adhesive frame. Immediately place a cover slip on top of the aros to create a flat surface and leave at room temperature for five to 10 minutes. Remove the cover slip for one to two minutes while the sample is harvested.
Harvest a one milliliter aliquot of the sample following centrifugation at 8, 000 RPM for the seconds decant the supernatant resuspend the pellet in 200 microliters of fresh medium. Take 2.5 microliters of the concentrated sample and pipette onto the pre-prepared aros pad. Spread the sample evenly along the flat surface of the aros pad.
Then remove the adhesive frame from the microscope slide using tweezers without touching the aros pad. Transfer the aros pad from the microscope. Slide to a 35 millimeter diameter Petri dish with a glass cover slip bottom.
For high resolution imaging. Invert the aros pad so that the cell suspension is in contact with the cover slip of the Petri dish. On the day of the experiment, turn on the OMX 3D SIM imaging system at least one hour prior to imaging to allow the system and lasers to fully stabilize.
Then turn on the different components as listed in the text protocol on the master controller workstation. Open the OMX software by clicking the icon. Set the file path for data saving to an appropriate data folder.
Next, start the soft work software. Turn on the lasers required for the experiments on the main screen in the light parameters section. Activate the FITC camera from the channel selection and proceed to check the parameters as listed in the text protocol.
Apply a drop of oil to the top of the objective. Place the sample Petri dish containing the prepared cells into the stage inset and cover with the circular heating lid. Lower the stage until the oil touches the bottom of the sample Petri dish.
Allow the dish to heat equilibrate for 15 minutes In the stage using a low exposure setting, focus on the sample using the nano positioning stage controls. Focus up and down through the sample to determine if the light is spread evenly above and below the sample focal plane. Then use 0.5 micron step sizes and mark the top and bottom of the image stack before returning to the middle of the sample in the experiment tab, click the get thickness button to set the sample acquisition thickness.
Determine the maximum intensity value of the sample image with the current exposure and percent of light transmitted settings. This value is found below the image window. For time-lapse imaging, adjust the exposure and percent of light transmitted to obtain a maximum intensity of 1100 to 1400.
Above background. For the image, activate the time lapse section in the experiment tab. Set the required frame rate, and the total time in data file.
Enter an appropriate file name. Click the run button to begin the image acquisition at the start of the image acquisition. Note the maximum intensity value of the image at the beginning of the image acquisition for the first frame of the time series.
At the end of the acquisition of the first frame, check that there has not been more than a 10%decrease in signal intensity through the Zack for this first frame. At the end of the acquisition of the time series, open the resulting raw image file with a DV suffix and ascertain the decrease in maximum signal intensity from the beginning of the time series to the final image. Discard any time points where there has been more than 30%photo bleaching from the process menu, activate the SI reconstruction window.
Use the preexisting settings for all of the parameters. Activate the used channel specific OTF button and set to the standard filter set. Activate the used channel specific KO angles button and set to the standard filter set.
Drag and drop the raw file into the input file space. Click do it. Examine the distribution of FTSZ around the Z ring to create 3D intensity plots.
Then open a 3D image file. To view the individual z slices. Use the volume viewer tool located under the view menu tab.
To crop a region of interest, enter the window number for this 3D image into the input window and enter a new and unused window number into the output window. The select region button will become available to crop an individual Z ring of interest from the original 40 by 40 micron field of view. Adjust the desired axis and degree of rotation in the rotation tab.
Click the do it button. Scroll through the volume to obtain the desired perspective, the Z ring. Use the data inspector tool to adjust the column row dimensions to create a new region of interest around an individual Z ring.
Click the center of the image to position this new region of interest. The text image data is an automatically generated table that displays the fluorescence intensity of each pixel within the region of interest. Click the 3D graph button to initially view the intensity plot.
Alternatively, the text image data can be exported by clicking the save table data button. In the file menu, copy the image data from the saved text file into a new spreadsheet. Select all the cells within the spreadsheet and create a new 3D surface graph.
Format the 3D intensity plot graph for publication for time-lapse data. Repeat these steps on each of the different time points from the 3D image file. When visualized by conventional widefield fluorescence microscopy, the Z ring appears as a single transverse band of fluorescence in bacillus subtles.
The fluorescence intensity throughout this band is uniform. The localization pattern offers little insight into the structural organization and distribution of FTS said polymers within the ring here, the same cells are examined. Using the FAST 3D sim method.
Rotation of the 3D sim image around the Z axis enables visualization of the Z ring as a genuine ring structure in three dimensional space. The improvement in resolution allows visualization of the heterogeneous distribution of FTSZ within the ring. Additional examples of Bacillus Z rings visualized by this technique illustrate how the fluorescence intensity around the Z ring is never the same.
Moreover, regions of very low fluorescence intensity or gaps are observed. To quantify these observations, 3D intensity plots were created of the Z ring. The intensity plots show that the amount of fluorescence can vary up to fourfold in different regions of the bacillus subtlest Z ring.
The 3D intensity plots show that the gaps almost read baseline levels of background fluorescence to analyze the dynamic changes that occur within the Z ring. Over time, 3D sim time-lapse is performed. This movie shows how FTSZ within the bacillus subtle Z ring is constantly changing and is measured with an intensity plot that is generated for each image.
After watching this video, you should have a good understanding of how to grow and prepare bacterial cells, how to image them using 3D Sim, how to analyze them to obtain quantitative data, and finally, how to provide presentation quality images. It's really important during image acquisition to monitor the health of your cells and also to monitor the level of the expression of the fluorescent protein during the acquisition After its development. This technique paved the way for other people in the field of bacterial cell biology to use other super resolution microscopy methods and to localize other bacterial proteins in different organisms.
Following this procedure, other forms of super resolution microscopy could be performed such as photo activated localization microscopy to have a look at dynamic changes in the FDS said protein over time.
