This protocol facilitates the direct observation of various phenotypic changes at a single-cell level and allows the continuous monitoring of protein localization and gene expression timing. The main advantage of fluorescence microscopy is that it is a straightforward method for monitoring various biological processes, such as cell division and cell morphology changes in live cells. Be sure to pay particular attention to the sample preparation protocol and to explore the microscope software to be able to locate the settings and options listed in this protocol.
A visual representation of this protocol is critical because it allows users to watch and learn the significant steps of the technique as they set up their own experiments. Begin by labeling a five to 50-microliter aliquot of the cell culture of interest with an appropriate membrane staining dye and adding five microliters of the stained bacterial cells onto a cover slip on the bottom of a 35-milliliter glass bottom culture dish. Place an 11-millimeter diameter agarose slab over the sample and gently tap to make sure the slab is flat against the cover slip.
Then add approximately five microliters of water around the cover slip to prevent the agarose pad from drying and allow the culture dish to equilibrate to the temperature inside the incubation chamber of a high-resolution deconvolution microscope for 15 to 20 minutes. To image the sample, use the coarse adjustment focus knob to make sure that the objective is fully lowered before initializing the microscope within the microscope software. Place a drop of 1.517 refractive index oil into the 100x oil immersion objective, and transfer the glass bottom dish containing the sample into the metal housing coffin.
Gently slide the dish into the stage clamp, and use the coarse adjustment knob to raise the objective until the oil makes contact with the glass bottom of the dish. Use the eyepiece and the fine adjustment knob to bring the sample into focus, and switch to Camera Mode. In the imaging software on the Resolve 3D window, select the Design/Run Experiment icon.
A new dialog box will appear entitled Design/Run Experiment. Open the Design and Sectioning tabs in the dialog box to set the number of Z stacks and the sample thickness. To measure the thickness of the cells in the sample, incrementally adjust the Z-plane using the up and down arrows in the Resolve 3D dialog box, making where the cells go out-of-focus as the upper and lower limits for the Image Acquisition.
After adjusting the Percent Transmission of the Light Intensity and the duration of the Exposure for the individual selected channels in the Resolve 3D dialog box, click Point List to open the Points List. In the Design and Time Lapse tabs, select the Time Lapse checkbox and enter the time lapse parameters. Select the Visit Point list option, and enter the points to be imaged in the text box, separating the points by commas or hyphens for complete sequences.
Then edit file names and file locations in the Run tab and select Play in the Begin Experiment dialog box to start the experiment. At the end of the analysis, open the RAW image files of interest in an appropriate deconvolution program, and select the Process tab and Deconvolve. Perform any manual background noise subtraction and brightness contrast adjustment in any or all wavelength channels as necessary before saving the image as a TIFF file.
For cell length quantification, open the deconvoluted image files in the manufacturer supplied software or in free imaging software, such as ImageJ, and open the Tool tab. Then select Measure Distances and left-click the start and end points on the desired image file to measure the cell length. For fluorescent signal quantification, select Data Inspector.
Draw a box with the Column/Row option set to the specific dimension of interest, and select the area with the signal to be quantified. The addition of xylose to induce a GGS 8 strain results in a six-cell phenotype. While empty vector controls appear similar to control cells grown in the absence of inducer.
Overproduction of staph aureus gpsB disrupts cell division in B.subtilis as measured by time lapse microscopy and cell length quantification. In addition, the localization patterns of FtsZ or one of the proteins associated with this protein can be used a reporter to study and/or identify novel antimicrobial compounds through the time lapse imaging and cell length quantification. Be sure to use an oil with the appropriate refractive index as the refractive index may change depending on the temperature used for the imaging.
Deconvolution can be performed if the user wishes to remove out-of-focus noise, and data analysis involving fluorescent signal quantification and cell length and width measurements can also be performed.
