A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.

New flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline

Transmission electron microscopy (TEM) is an important imaging technique in bacterial research and requires ultrathin sectioning of resin embedding of cell pellets. This method consumes milli- to deciliters of culture and results in sections of randomly orientated cells. For rod-shaped bacteria, this makes it exceedingly difficult to find longitudinally cut cells, which precludes large-scale quantification of morphological phenotypes. Here, we describe a new fixation method using either thin agarose layers or carbon-coated glass surfaces that enables flat embedding of bacteria. This technique allows for the observation of thousands of longitudinally cut rod-shaped cells per single section and requires only microliter culture volumes. We successfully applied this technique to Gram-positive Bacillus subtilis, Gram-negative Escherichia coli, the tuberculosis vaccine strain Mycobacterium bovis BCG, and the cell wall-lacking mycoplasma Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we examined cellular changes induced by a panel of different antibiotics. Surprisingly, we found that the ribosome inhibitor tetracycline causes significant deformations of the cell membrane. Further investigations showed that the presence of tetracycline in the cell membrane changes membrane organization and affects the peripheral membrane proteins MinD, MinC, and MreB, which are important for regulation of cell division and elongation. Importantly, we could show that this effect is not the result of ribosome inhibition but is a secondary antibacterial activity of tetracycline that has defied discovery for more than 50 years.SignificanceBacterial antibiotic resistance is a serious public health problem and novel antibiotics are urgently needed. Before a new antibiotic can be brought to the clinic, its antibacterial mechanism needs to be elucidated. Transmission electron microscopy is an important tool to investigate these mechanisms. We developed a flat embedding method that enables examination of many more bacterial cells than classical protocols, enabling large-scale quantification of phenotypic changes. Flat embedding can be adapted to most growth conditions and microbial species and can be employed in a wide variety of microbiological research fields. Using this technique, we show that even well-established antibiotics like tetracycline can have unknown additional antibacterial activities, demonstrating how flat embedding can contribute to finding new antibiotic mechanisms.

From Light Microscopy to Analytical Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB)/SEM in Biology: Fixed Coordinates, Flat Embedding, Absolute References

Correlative light and electron microscopy (CLEM) has been in use for several years, however it has remained a costly method with difficult sample preparation. Here, we report a series of technical improvements developed for precise and cost-effective correlative light and scanning electron microscopy (SEM) and focused ion beam (FIB)/SEM microscopy of single cells, as well as large tissue sections. Customized coordinate systems for both slides and coverslips were established for thin and ultra-thin embedding of a wide range of biological specimens. Immobilization of biological samples was examined with a variety of adhesives. For histological sections, a filter system for flat embedding was developed. We validated ultra-thin embedding on laser marked slides for efficient, high-resolution CLEM. Target cells can be re-located within minutes in SEM without protracted searching and correlative investigations were reduced to a minimum of preparation steps, while still reaching highest resolution. The FIB/SEM milling procedure is facilitated and significantly accelerated as: (i) milling a ramp becomes needless, (ii) significant re-deposition of milled material does not occur; and (iii) charging effects are markedly reduced. By optimizing all technical parameters FIB/SEM stacks with 2 nm iso-voxels were achieved over thousands of sections, in a wide range of biological samples.

Global embeddings and hydrodynamic properties of Kerr black hole

In the presence of a rotating Kerr black hole, we investigate hydrodynamics of the massive particles and massless photons to construct relations among number density, pressure and internal energy density of the massive particles and photons around the rotating Kerr black hole and to study an accretion onto the black hole. On equatorial plane of the Kerr black hole, we investigate the bound orbits of the massive particles and photons around the black hole to produce their radial, azimuthal and precession frequencies. With these frequencies, we study the black holes GRO J1655-40 and 4U 1543-47 to explicitly obtain the radial, azimuthal and precession frequencies of the massive particles in the flow of perfect fluid. We next consider the massive particles in the stable circular orbit of radius of 1.0 ly around the supernovas SN 1979C, SN 1987A and SN 2213-1745 in the Kerr curved spacetime, and around the potential supermassive Schwarzschild black holes M87, NGC 3115, NGC 4594, NGC 3377, NGC 4258, M31, M32 and Galatic center, to estimate their radial and azimuthal frequencies, which are shown to be the same results as those in no precession motion. The photon unstable orbit is also discussed in terms of the impact parameter of the photon trajectory. Finally, on the equatorial plane of the Kerr black hole, we construct the global flat embedding structures possessing (9 + 3) dimensionalities outside and inside the event horizon of the rotating Kerr black hole. Moreover, on the plane, we investigate the warp products of the Kerr spacetime.

Local free-fall temperature of modified Schwarzschild black hole in rainbow spacetime

We obtain a (5+1)-dimensional global flat embedding of modified Schwarzschild black hole in rainbow gravity. We show that local free-fall temperature in rainbow gravity, which depends on different energy [Formula: see text] of a test particle, is finite at the event horizon for a freely falling observer, while local temperature is divergent at the event horizon for a fiducial observer. Moreover, these temperatures in rainbow gravity satisfy similar relations to those of the Schwarzschild black hole except the overall factor [Formula: see text], which plays a key role of rainbow functions in this embedding approach.