This method can help answer key questions in the field of microbiology, such as how DNA structure changes during the cell cycle of bacteria. The main advantage of this technique is that it is possible to visualize ultrastructure of whole bacterial cells close to the living state at appropriate time points without any additional treatments. Demonstrating the procedure will be Chihong Song, a postdoc from my lab, and Mako Hayashi, a grad student from Dr.Kaneko's lab.
Begin with growing cyanobacteria on nine-centimeter, sterilized BG-11 plates containing 1.5%agar and 0.3%sodium thiosulfate. Incubate the plates at 23 degrees Celsius under a 12-12 light cycle with 50 micro-E of light per square meter per second. To maintain the cells, transfer them onto fresh BG-11 agar plates weekly.
Within one week, cultures appear as green bands. Transfer the green clumps of cells using a flame-sterilized loop, and streak them onto the new plate. To observe DNA compaction, collect the cells cultured for six days at the end of the light period.
To release the cells from the plate, add one milliliter of 0.2-molar sucrose. Then, collect the cell suspension, and repeat the sucrose addition and removal until the solution turns green. The color change indicates that most cells have been released.
Now, transfer 500 microliters of suspended-cell solution to a Microfuge tube, and add Hoechst stain for a final concentration of one microgram per milliliter. Then, let the cells incubate in the dark for 10 minutes. Next, spin down the cells, discard the supernatant, and resuspend them in 10 microliters of 0.2-molar sucrose.
Next, transfer one microliter of the suspension to a glass slide, put on a cover slip, and observe the cells under a fluorescence microscope equipped with a UV filter. Using a 100 times oil immersion objective, look for evidence of DNA compaction, which should be observable in most of the cells. First, prepare the plunge-freezing device.
Then, set up the liquid nitrogen container by inserting the ethane container and cooling rod attachment. Next, fill the container with liquid nitrogen, and cool it to liquid nitrogen temperature. After that, load ethane gas to the container.
Be sure to wear safety glasses, as liquid ethane is explosive. Finally, remove the cooling rod attachment, and cover the container with a plastic lid to avoid attaching frost. To proceed, glow discharge the carbon side of a carbon-coated EM grid for 30 seconds at 50 milliamps using a plasma ion barrier.
Then, prepare the Vitrobot according to the test protocol. Use tweezers to set the pretreated EM grid to the chamber. Treat the EM grid with one microliter, 15-nanometer BSA gold tracer to serve as a fiducial marker before applying the sample.
Now, aliquot 2.5 microliters of cell suspension onto the grid. Blot off any excess solution using filter paper, and immediately freeze the grid in liquid ethane. Keep the frozen grid stored in liquid nitrogen until they can be examined.
Next, start up the HVEM, and set it to a high voltage of one megavolt. Then, cool the specimen grid holder to minus 150 degrees Celsius using liquid nitrogen. Once cooled, mount the frozen grid into the cooled cryo-specimen holder, and load it into the HVEM.
Take care to avoid contaminating the setup with ice. Now, select an imaging area at the low magnification of 1, 000 times. Then, adjust the eucentric z-axis height, and tilt the specimen stage to negative 60 degrees.
Then, remove the backlash of the tilting rotation. Now, focus near the target location at a magnification of 10, 000 times. Set an under focus of six to 10 microns by deviation from the focused image.
For imaging, set the dose to two electrons per square angstrom per second or less. Keep an eye on the electron dose, which is reflected by the current density, and take an image by digital camera or on an electron film. Next, collect tilt images from negative 60 to positive 60 degrees at increments of two to four degrees.
Use the automated features of the microscope if possible. Open the commercial software package, and load the tilt images for the tomographic reconstruction. Then, make an image stack file from the individual images using the command tif2mrc or newstack.
Next, start the eTomo GUI software, and input the image parameters for pixel size, fiducial diameter, image rotation, and so forth. Thus, create the editing script. Now, use the suggested software to create a tilt series, aligned using fiducial markers, with a mean residual error of less than 0.5.
Finally, reconstruct a 3D tomogram using the SIRT algorithm. Now, extract a region of interest from the tomogram, and apply a denoising filter, such as an anisotropic diffusion filter, a bilateral filter, or a mathematical morphology filter. Perform the filtering using parameters that enhance the image contrast.
Next, segment a feature of interest. Use the slicing feature to open the tomogram. Then, go to the Segmentation Editor window, and select a new label field to create a segmentation file.
Then, manually trace the border of the feature of interest in the first slice and then each of the other slices. Additional feature of interest can be added using the same operations. Now, to generate a surface rendering using options in the SurfaceGen menu to visualize the segmented volume, select the SurfaceView menu.
To move, rotate, and zoom in the 3D volume, use the tools in the 3D Viewer window. Automatic segmentation can be done using the Magic Wand Tool. Click on an object, and adjust the sliders in Display and Masking so that the features of the object are fully selected.
In a precise synchronous culture, DNA labeled with Hoechst shows a normal uniform distribution in the dark condition and then progressively compacts within the cell during the light period, taking on a wavy rod-like structure by the end of the light period. Finally, the rod divides at the center, and its two parts are distributed into the daughter cells. After the cell division, the compacted DNA disappears immediately, and the DNA returns to a normal uniform distribution.
When cells in the final stage of DNA compaction were frozen and observed using high-voltage electron microscopy, many showed distinct DNA compaction that was easily distinguished from normal cells. Some exhibited a constriction at the center of the cells, as expected before cell division. From the 3D tomograms, compact DNA is visibly separated in the cytoplasm and is surrounded by a low-density material.
The thylakoid membrane layers are distorted along the wavy rod of compacted DNA. Interestingly, many small phosphate bodies adhere to the compacted DNA and usually appear in pairs. These polyphosphate bodies may be the suppliers of phosphate for DNA synthesis.
After watching this video, you should have a good understanding of how to visualize ultrastructure of whole bacterial cells close to the living state at the appropriate time point by cryo-high voltage electron microscopy without any additional treatment like staining. While attempting this procedure, it is important to remember that the state of DNA compaction changes with each minute at the end of the light cycle. Make sure you don't miss the best timing to freeze the sample.
Following this procedure, other methods like CLEM can be performed in order to answer additional questions about the localization of specific proteins or nucleotides in the compacted DNA. After its development, this technique should pave the way for researchers in the field of microbiology to explore cell division, DNA segregation, and viral infection in bacteria or in small eukaryotes.
