Gene Expression in Electron-Beam-Irradiated Bacteria in Reply to “Live Cell Electron Microscopy Is Probably Impossible” Eamonn Kennedy,† Edward M. Nelson,† John Damiano,‡ and Gregory Timp*,§ †
Department of Electrical Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States Protochips, Inc., Morrisville, North Carolina 27560, United States § Departments of Electrical Engineering and Biological Science, University of Notre Dame, Notre Dame, Indiana 46556, United States ‡
n a recent letter entitled “Live Cell Electron Microscopy Is Probably Impossible” Niels de Jonge and Diana B. Peckys1 criticized our paper on live-cell imaging2 as unrealistic, even though the visualization of Escherichia coli was accomplished in a liquid cell using low-dose scanning transmission electron microscopy. Their criticism hinged on two claims: first, that the minimum electron dose to obtain contrast for scanning transmission electron microscopic (STEM) visualization was “many orders of magnitude above the lethal dose”; and second, that our interpretation of the viability assay was faulty. In this rebuttal, we argue, to the contrary, that it is entirely realistic to visualize E. coli within the unforgiving range of parameters tested so far. Our argument has five salient aspects. First, de Jonge and Peckys’ argument regarding the minimum electron dose stemmed from a flawed premise, originating with Reimer and Kohl.3 Although their reference was not specific, we assume that de Jonge and Peckys were referring to the discussion of Table 11.4 in the book by Reimer and Kohl where it was remarked that reproductive death of E. coli was supposed to occur near q = 8 × 10−8 C/cm2, associated with a dose of a 0.005 e−/nm2 at 100 kV. (Likewise, in the citation attributed to Isaacson,4 Table 1.7 referred to radiation damage measured by the colony-forming properties of E. coli at a dose of 6.2 × 10−4 e−/nm2.) Reimer and Kohl went on to estimate the minimum dose required for a high-resolution (d = 5 nm) image of a biological specimen to be about qmin = ek2/fd2C2 ∼ 3 × 10−4 C/ cm2 = 37 e−/nm2, where κ = 1 (κ = 5 is the Rose criterion) denotes the signal-to-noise ratio, C = 0.01 represents the contrast, and f = 1 is the fraction of electronics contributing to the image background, which seems to be orders of magnitude above the estimated lethal dose. The premise of de Jonge and Peckys’ letter was essentially flawed because the values offered by Reimer and Kohl (and Isaacson) were scaled; they were interpretations of results, not measurements, obtained from a linear conversion of absorption measurements in water (generally) taken under very different circumstances (e.g., with 70 kV X-rays or 1 to 50 MeV electron beams)5 from the imaging accomplished in our work. The rationale used for this interpretation relied on a translation of “Rads” or Grays associated with absorption to an imaging dose, but it ignored the differences between absorption and imaging. Unlike absorption, only a small f raction of the beam energy is actually transferred to the thin specimen during imaging. This caveat was stipulated explicitly by Isaacson and others.4,6 Thus,
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the interpretation offered in Reimer and Kohl cannot be compared directly to the doses used in our work. However, there have been measurements7,8 using TEM to image E. coli in a liquid cell at 200 kV that report a lower bound on the lethal dose, indicating only that E. coli remained viable for doses >5 × 10−6 e−/nm2 at 200 kV, according to a LIVE/DEAD Baclight viability assay. Second, de Jonge and Peckys mistakenly represent that the prokaryotic cells (bacteria) in our study cannot be viable after electron-beam exposure because the “...minimum dose used was 0.2 e−/Å2 (20 e−/nm2).” Contrary to their representation, the first micrograph presented in the paper was acquired using 4 e−/nm2 (Figure 1d in the original article). Furthermore, Figure 1f in the original paper conveys a monotonically decreasing viability from 1 e−/nm2 with increasing dose, which is 20-fold below the minimum electron dose they attribute to our work in their letter. Parenthetically, the images shown in Figure 3a−d in the original article were all acquired at doses less than or comparable to 20 e−/nm2. Third, their argument about viability after electron irradiation is immaterial as it proceeds from a misleading comparison between the data we acquired exclusively from strains of E. coli, a prokaryote and one of the most widely used bacterium in genetic engineering and biotechnology, and their results from yeast, the simplest of eukaryotic cells. Among the myriad of differences between yeast and E. coli, what is especially relevant is that the cell wall in yeast, which encapsulates a volume of about 50 μm3 including the nucleus, cytoskeleton, and subcellular organelles, consists of a microfibrillar array of glucan chains with a laminated proteoglycan layer that is estimated to be about 105−115 nm thick, depending on how it is measured.9,10 Unlike yeast, E. coli have no nucleus and are characterized by a cell envelope encapsulating about 1 μm3 that consists of an inner cytoplasmic (CM) and outer (OM) cell membrane with a thin peptidoglycan sacculus, which is essentially one giant macromolecule, within an aqueous compartment (the periplasm) sandwiched in between.11,12 As evident from Figure 2b in the original article,2 the OM and CM were estimated to be about 8−18 nm thick, separated by about 34 nm. Parenthetically, the elasticity of the cell envelope (attributed mainly to the sacculus) is supposed to be 2.5 MPa,13 Received: September 30, 2016 Published: January 24, 2017 3
DOI: 10.1021/acsnano.6b06616 ACS Nano 2017, 11, 3−7
Letter to the Editor
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Figure 1. E. coli viability measured by gene expression after electron irradiation. (a) A plasmid map of pNFK-113, which is denoted as “113“in the text, with degradable versions of LuxI and GFP is shown. (b−d) Top: Transmission optical micrographs of the liquid cell are shown prior to electron-beam irradiation (left), and 10 min (middle) and 90 min (right) after exposure to an electron dose of 20 e−/nm2 and subsequent induction with 700 μM concentration of IPTG. Middle: Magnified views of the same transmission optical micrographs shown in (b−d, top). Bottom: The flurorescence micrographs corresponding to (b−d, middle), acquired from the liquid cell, illustrating both live and dead E. coli. Only 6 out of 16 113 E. coli shown are expressing GFP-LVA. (e) 113 E. coli viability, measured by GFP-LVA expression 120 min after induction by IPTG (solid circles) and Baclight staining (open circles), is shown as a function of the mean electron-beam dose. Inset: Time dependence of the cumulative fluorescence per cell, illustrating that the fluorescence after IPTG induction increases at about the same rate with (red, 20 e−/nm2) and without (blue) electron-beam irradiation, although the gene expression diminishes after irradiation.
about 4-fold stiffer than yeast.14 Taken together, the cell volume, which affects absorption, and the differences between membrane thickness, architecture, composition, and stiffness confound a direct comparison between yeast and E. coli.
Fourth, de Jonge and Peckys discounted our estimate of the lethal dose sufficient to kill 50% of the population, LD50, because it relied on a LIVE/DEAD Baclight assay for measuring (bacterial) viability. Our reliance on the LIVE/DEAD assay 4
DOI: 10.1021/acsnano.6b06616 ACS Nano 2017, 11, 3−7
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followed a plethora of other work using the same test,7,8,15 and it was based on the observation that with necrosis and/or apoptosis, the cell’s metabolism shuts down; it loses membrane integrity; and it lyses. Thus, membrane integrity is routinely used as a measure of viability. Briefly, the assay uses a membrane-permeant SYTO 9 label to report live bacteria with green fluorescence, and membrane-impermeant propidium iodide (PI) to identify membrane-compromised bacteria with red fluorescence. SYTO 9 penetrates the bacterial cell membrane and binds to nucleic acids in both live and dead cells, whereas PI penetrates and intercalates into nucleic acids only if the membrane is compromised (i.e., when a cell dies). When SYTO 9 and PI are mixed and applied to a dead cell, PI preferentially binds to nucleic acids, such that SYTO 9 (green) fluorescence weakens and PI (red) fluorescence predominates. Although several factors have to be taken into account, such as different binding affinities to live and dead cells and bleaching of SYTO 9,16 the interpretation of the data was unambiguous so long as both the red, green, and background fluorescence channels were accurately recorded.2 The results of a LIVE/DEAD assay, performed as a function of electron-beam dose at 300 kV, revealed that LD50 = 29.4 e−/nm2, which coincidently was in-line with other work showing a loss of membrane integrity there (Figure 1f in the original article).17 Importantly, Figure 1f conveyed a monotonically decreasing viability with increasing dose delivered to the cell, starting near 80% viability at 1 e−/nm2 and decreasing below 10% at 80 e−/ nm2. Since, according to de Jonge and Peckys, all these cells should be dead regardless as the dose was “many orders of magnitude above the lethal dose”, the membrane integrity should have been compromised, too, which was clearly not the case, refuting the premise of their letter. Instead, the data support the conclusion that the cells were alive but increasingly compromised by doses above 29 e−/nm2. In agreement with this assessment, but in an apparent contradiction to their letter, de Jonge and Peckys indicated in a recent publication that a “snapshot” acquired with liquid STEM using a dose of 40 e−/ nm2 informed on the ultrastructure of a live cell as assessed by fluorescence microscopy, and that subsequent images in other areas contained information about the live cell (although damage may have occurred).18 Admittedly, the LIVE/DEAD assay was an imperfect measure of E. coli viability. Even if the membrane remained intact, radiation damage was inescapable because secondary effects such as the production of small diffusible radicals like OH− in a hydrated specimen were likely to occur,19 affecting life processes. However, the observations of binary fission, the low-frequency tumbling of E. coli under the beam, and the infection of E. coli by P1 bacteriophage, also reported in the original article, lent further support to the idea that the cells were still alive. For example, motile (tumbling) bacteria were reportedly observed at low electron doses (see video V1 in the original article, Supporting Information), but the motion stopped within seconds for >10 e−/nm2·frame electron doses, indicating the adverse effect of a high dose. Another example illustrating the dynamics of a life process was the infection of E. coli by P1, which was the main focus of our paper. The infection process was supposed to involve, first, absorption of the phage to receptors, the terminal glucose residue in the core of the lipopolysaccharide molecules present on the outer surface of the OM of the host cell, and then (contraction of the tail and) injection of the viral DNA, which was actually visualized in Figure 3d of the original article,
although only a fraction (typically
