This protocol describes how to visualize the transient DNA compaction in cyanobacteria. Synchronous cultivation, monitoring by fluorescence microscopy, rapid freezing, and high voltage cryo-electron tomography are used. A protocol for these methodologies is presented, and future applications and developments are discussed.
This protocol describes how to visualize the transient DNA compaction in cyanobacteria. DNA compaction is a dramatic cytoplasmic event recently found to occur in some cyanobacteria before cell division. However, due to the large cell size and the transient character, it is difficult to investigate the structure in detail. To overcome the difficulties, first, DNA compaction is reproducibly produced in the cyanobacterium Synechococcus elongatus PCC 7942 by synchronous culture using 12 h each light/dark cycle. Second, DNA compaction is monitored by fluorescence microscopy and captured by rapid freezing. Third, the detailed structure of DNA compacted cells is visualized in three dimensions (3D) by high voltage cryo-electron tomography. This set of methods is widely applicable to investigate transient structures in bacteria, e.g. cell division, chromosome segregation, phage infection etc., which are monitored by fluorescence microscopy and directly visualized by cryo-electron tomography at appropriate time points.
DNA compaction is a dramatic cytoplasmic event that has been identified in some cyanobacteria. When Synechococcus elongatus was cultured under 12 h each light/dark cycle, DNA appeared condensed at the end of the light period, which was clearly different from its appearance at the other time points1. It has been suggested that this process is controlled by a circadian clock based on the Kai proteins2. Seki et al. have reported that the DNA stained with Hoechst 33342 was compacted in S. elongatus cells toward the end of the light period and showed a wavy rod-shape under a fluorescence microscope. The compacted DNA then separated into two at the center of the rod as the cell divided, and finally returned to a normal uniform distribution in each daughter cell3. However, its transient nature and large size for electron microscopy impeded structural analysis. Murata et al. combined several methods, including synchronous culture, fluorescence microscopy, rapid freezing, and high voltage electron cryo-tomography (cryo-HVET), and succeeded in identifying the structure of transient DNA compaction, including the kinetics of polyphosphate bodies (PPBs)4. The manuscript provides visual explanation of such a difficult material in detail by combining the experimental procedures.
S. elongatus has a capsule shape, a length of 2 to 5 µm, a width of about 0.5 µm, and the perfect DNA compaction appears in living cells only for a very short time. Therefore, the structural changes occurring in the cyanobacterial DNA compaction were unknown in detail. In order to investigate these structures by electron microscopy, it is necessary to overcome two main technical problems. One is the observation of such a thick specimen of the whole bacterium at near native conditions, and the other is the rapid fixation of a dynamic structure. As for the first problem, the inelastic mean free path (iMFP) of electrons depends on the accelerating voltage of the electron microscope5. In a transmission electron microscope (TEM) of 300 kV, it is less than 350 nm. For example, when an ice-embedded cyanobacterium (specimen thickness ≈ 600 nm) is observed in 200kV TEM (iMFP ≈ 250 nm), the structures inside cell are difficult to observe. By contrast, 1 MV TEM (iMFP ≈ 500 nm) can give and image of the cytoplasmic structure throughout the cell (Figure 1). In this protocol, as one part of the solution, a high voltage electron microscope (HVEM) at an accelerating voltage of 1 MV was employed. However, facilities that implement HVEM are limited worldwide. Possible alternative solutions are also discussed in the Discussion section. The second problem was solved by cryo-electron microscopy (cryo-EM). This is a powerful tool for visualizing dynamic structures at near native conditions, where the specimen is rapidly frozen in liquid ethane using a rapid freezing device, and the frozen moment is directly observed with a minimum of modifications6. Combining with tomography, a snapshot of three-dimensional (3D) structures can be reconstructed from the tilt series7. In this experiment, DNA compaction was reproduced in S. elongatus using synchronous culture under 12 h each light/dark cycle, and the timing of the freezing of the specimen was determined by monitoring under a fluorescence microscope.
The approaches described here are widely applicable to the study of dynamic structures in bacterial cells, e.g. cell division, chromosome segregation and phage infection, and have a potential to open new avenues in microbiological research.
We have presented a sequence of protocols for visualizing transient DNA compaction in cyanobacteria. The basic concept is similar to that of correlative light and electron microscopy (CLEM)12. In addition, in this method, live cyanobacteria were monitored by fluorescence microscopy, rapidly frozen on EM grids, and directly visualized with high voltage cryo-electron tomography. As a first application, the detailed structure of DNA compacted bacterial cells was successfully visualized in 3D. Currently, this procedure is specific to this subject, but it will be applied more extensively, with modified methodology in some cases. Here, the advantages, the limitations, and the future possibilities of this method are discussed.
One of the advantages of this method is the 3D visualization of the whole cell. 1 MV HVEM successfully visualized the dynamic structure of the subcellular organelles in the DNA compacted cells. However, the fine structure inside normal cells could not be distinguished due to low image contrast. Increasing inelastic and multiple scattering in thick specimens blurs the image13. Zero-loss and most-probable-loss image filtering by an energy filter can improve image contrast by reducing inelastic scattering14,15, but it will not work for specimens thicker than iMFP. The zero-loss and most-probable-loss peaks drastically decrease with specimen thickness. It is particularly difficult to obtain a sufficient signal-to-noise ratio for the electron sensitive ice-embedded specimens. Murata et al. have shown that 1MV scanning transmission microscopy (STEM) gives higher image contrast than a bright field image in plastic embedded yeast cells with 5 µm thickness, where the image contrast is primarily given by amplitude contrast13. However, it is expected that the effect of knock-on damage on higher accelerated electrons creates another limitation to the irradiation dose for damage-sensitive cryo-specimens16. The application of Volta and Zernike phase plates17,18 for HVEM may be able to reduce knock-on damage by reducing the total dose in the future. Another limitation to using HVEM for thick specimens comes from the fact that the user facilities that provide HVEM are scarce worldwide.
Using an alternative methodology to observe thick specimens, cryo-STEM tomography at 300 kV has demonstrated high contrast images on frozen-hydrated specimens with thickness in excess of several hundred nanometers19. To retrieve phase contrast in cryo-STEM, electron pthychographic microscopy has also been introduced, in which the phase plate in the condenser lens transposes a phase-modulated diffraction to a pixelated 2D detector20. The images are retrieved by calculation from multiple diffractions. For direct and fast 3D cryo-imaging of large native frozen samples, cryo-FIB-SEM can also be used21, where serial sectioning with a focused ion beam and block face imaging is applied for imaging fully hydrated frozen specimens. Although these technologies expand the viewing range of biological specimens, it is difficult to find the target location of the bacteria, e.g. labeled bacteria, because the target is completely under the ice and cannot be identified before trimming.
DNA compaction produces a distinct structure in cyanobacteria. DNA compacted cells are readily distinguished even without being stained due to a large density bias within the cells that is not present in normal cells. However, in order to visualize more local events within the cell, it is necessary to transfer the fluorescently labeled ROI into the electron microscope. For correlative light and electron microscopy (CLEM), light microscope images and electron microscope images are generally correlated using fluorescent latex beads or quantum dots on EM finder grids12. The labelling particles must be of high electron density in addition to fluorescence. They can accurately and reliably correlate the positions between the two images. Furthermore, by confirming the labeled area with cryo-light microscopy, complete overlap of ROI can be achieved between the two microscopes. When characterizing more detailed structural events in DNA compaction, these particles and cryo-light microscope will be an indispensable tool for a more robust and accurate correlation in the future.
This article shows how to characterize the transient structure of DNA compaction in cyanobacteria by a combination of synchronous culture, fluorescence microscopy and high voltage cryo-electron tomography. This protocol focuses on the observation of compacted DNA. By combining this method with other new technologies mentioned above, it will be possible to investigate the process of DNA compaction in further detail, and suitably modified methods are widely applicable to other dynamic structural events in bacteria.
The authors have nothing to disclose.
The authors thank Tammo Reisewitz for critical reading of the manuscript, and both Mako Hayashi and Sayuri Hagiwara for careful cultivation and observation of cyanobacteria, Yoshitaka Kimori for image processing, Chihong Song, Naoyuki Miyazaki, and Miyoko Nagayoshi for helping with segmentation of the structures. This work was supported by the Collaborative Study Program of the National Institute for Physiological Sciences (NIPS) to Y.K.
Hoechst 33342 solution | Dojindo | 346-07951 | 1mg/mL in H2O |
Agar Powder (for plant culture) | Wako | 016-11875 | |
Boric acid | Wako | 027-02192 | 99.5% |
Manganese chloride tetrahydrate | Wako | 139-00722 | 99% |
Zinc sulfate heptahydrate | Wako | 264-00405 | 99.5% |
Sodium molybdate | Wako | 196-02472 | 99% |
Copper sulfate pentahydrate | Wako | 039-04412 | 99.5% |
Cobalt nitrate hexahydrate | Wako | 031-03752 | 99.5% |
Sodium nitrate | Wako | 191-02542 | 99% |
Magnesium sulfate heptahydrate | Wako | 131-00405 | 99.5% |
Calcium chloride dehydrate | Wako | 031-00435 | 99.5% |
Citric acid | Wako | 036-05522 | 98% |
EDTA-2Na | Dojindo | 343-01861 | 99.5% |
Sodium carbonate | Wako | 197-01581 | 99.8% |
Potassium phosphate dibasic | Wako | 164-04295 | 99% |
TES (Good’s buffer) | Dojindo | 344-02653 | 99% |
Ferric ammonium citrate | Wako | 092-00802 | 1st Grade |
Sodium thiosulfate pentahydrate | Wako | 197-03585 | 99% |
BSA gold tracer 15nm | Aurion | 215.133 | |
Quantifoil EM grid | Quantifoil MicroTools | R3.5/1 Copper grid | |
Electron films | Kodak | SO-163 | |
Developer | Kodak | D19 | |
Fixer | Kodak | Rapid fixer | Solution |
Filter paper | Whatman | Grade 1 | |
Growth chamber | NKsystem | LH-100SP | |
Fluorescent microscope | Nikon | ECLIPSE 50i | |
High voltage TEM | HItachi | H1250M | |
Cryo-specimen holder for HVEM | Gatan | ||
plunge-freezing device | Leica | EM CPC | |
Plasma Ion bombarder | Vacuum device | PIB-10 | |
Liquid nitrogen storage | Taylor-Wharton | 25LDB | |
Developing tank | Dosaka EM | TB-3-75 | |
flatbed scanner | Nikon | Coolscan 9000ED | |
Segmentation software | FEI | Amira | https://www.fei.com/software/amira |
Tomographic Reconstruction software | eTOMO | http://bio3d.colorado.edu/imod |