Passive response to hyperosmotic shock involves two phases of cell shrinking.

<p>(A) A sequence of fluorescent images of <i>E.coli</i> cells prior and during the hyperosmotic shock. Top: cytoplasmic volume marked with the green protein EGFP; Middle: total cell volume marked by the red outer membrane dye FM4-64; Bottom: overlay if EGFP and FM4-64. A cell is transferred from isoosmotic buffer (10 mM Tris-HCl buffer supplemented with 150 mM sucrose) to isoosmotic buffer and 620 mM sucrose. Images were acquired at a frame every 1.6 seconds alternating between the cytoplasmic volume and total cell volume. Image brightness was adjusted due to photo bleaching and the difference in fluorescence intensity between the FM4-64 and EGFP. Videos are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035205#pone.0035205.s003" target="_blank">Video S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035205#pone.0035205.s004" target="_blank">S2</a>. (B) Volume recovery versus time obtained using cell area analysis. Inset: cell length and cell radius versus time. Total cell volume, length and radius are given in red and cytoplasmic cell volume, length and radius in green. An example contour of the cytoplasmic cell volume and total cell volume obtained after cell area analysis for the initial frame is shown on the bottom left. The inset shows a cartoon illustration of the post sucrose shock events described in the text.</p

Slow recovery requires protein synthesis.

<p>(A)–(E) Averaged recovery traces shown for different shock magnitudes (V_n(t)). For each shock, the recovery trace in the presence (red) and absence (black) of the drug chloramphenicol is shown. Data was recorded and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035205#pone-0035205-g003" target="_blank">Figure 3</a>. 0.44 Osmol/kg (no shock, N = 10), 0.6 Osmol/kg (N = 5), 0.8 Osmol/kg (N = 5), 1.15 Osmol/kg (N = 20) and 1.45 Osmol/kg (N = 10). (F) Final volume, calculated as the average of the last 15 minutes of data in (A–E), versus shock magnitude. Error bars are standard error of the mean.</p

Volume recovery is complex and required for growth.

<p>Averaged recovery traces shown for different shock magnitudes (V_n(t)). The initial 10 minutes of data is recorded at 1 Hz frame rate, subsequent data is recorded at a frame every 30 seconds. For each cell in a given data set, the recovery trace was normalized by the initial volume, aligned at the time of hyperosmotic shock and resampled every 30 seconds. The average trace was computed from these normalized and aligned data sets. The following conditions are shown: 0.44 Osmol/kg data (N = 9), 0.5 Osmol/kg (N = 4), 0.6 Osmol/kg (N = 22), 0.8 Osmol/kg (N = 6), 1.15 Osmol/kg (N = 28), 1.45 Osmol/kg (N = 16), 2 Osmol/kg (N = 12) and 2.5 Osmol/kg (N = 5).</p

2D Probability density function (PDF) of the recovery traces for a given shock magnitude.

<p>Approximately 100 cells per shock magnitude are includes. PDFs are calculated from normalized and aligned data sets as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035205#pone-0035205-g003" target="_blank">Figure 3</a>. Cells were shocked by transferring them from LB into LB and a given amount of sucrose. Post shock osmolality is given in each panel.</p

Polynomial extrapolation of relative focal shift as a function of NA and <i>n</i>₂.

<p>(A) <i>α</i>_<i>poly</i> as a function of NA and <i>n</i>₂. The colormap (inset) gives the value of <i>α</i>. The TIR region is marked by a dashed gold line. Colored arrowheads indicate where the 1D slices through this surface are taken for panels C and D. (B) The order of the polynomial extrapolation is chosen as the lowest order whose maximal error (grape) is less than the maximal difference between <i>α</i>₆₄₆ and <i>α</i>₄₈₉ (cocoa). (C-D) <i>α</i> evaluated by the theory in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134616#pone.0134616.ref002" target="_blank">2</a>] at various values of NA and <i>n</i>₂ (symbols) along with the global fit to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134616#pone.0134616.e003" target="_blank">Eq 3</a> (lines). Symbols that corresponding to TIR systems are shown as faded circles. (C) <i>α</i> as a function of refractive index for three different values of numerical aperture (1.00 in turquoise, 1.38 in olive, 1.48 in orchid). Also included is the <i>n</i>₂/<i>n</i>₁ approximation (gray—⋅⋅). (D) <i>α</i> as a function of numerical aperture for three different values of <i>n</i>₂ (1.33 in pistachio, 1.38 in goldenrod, 1.43 in periwinkle). (E) Experimentally observed <i>α</i> for imaging system of varying refractive index. Refractive index of the medium was increased by including glycerol (cornflower) or sucrose (dark red). Two copies of the same model of objective were measured, results from one are filled symbols and the other are open. Evaluating the <i>α</i>_<i>poly</i> with an NA of 1.49 is shown in charcoal. <i>α</i> error bars are 90% CI. <i>n</i>₂ error bars reflect changes in additive concentration by ± 1.5%. (F) Experimentally observed <i>α</i>_{<i>multiBead</i>} as a function of NA for imaging systems with various objectives using the multiple bead sizes method.</p

Simple Experimental Methods for Determining the Apparent Focal Shift in a Microscope System

<div><p>Three-dimensional optical microscopy is often complicated by a refractive index mismatch between the sample and objective lens. This mismatch causes focal shift, a difference between sample motion and focal-plane motion, that hinders the accuracy of 3D reconstructions. We present two methods for measuring focal shift using fluorescent beads of different sizes and ring-stained fluorescent beads. These simple methods are applicable to most situations, including total internal reflection objectives and samples very close to the interface. For distances 0–1.5 <i>μ</i>m into an aqueous environment, our 1.49-NA objective has a relative focal shift of 0.57 ± 0.02, significantly smaller than the simple <i>n</i>₂/<i>n</i>₁ approximation of 0.88. We also expand on a previous sub-critical angle theory by means of a simple polynomial extrapolation. We test the validity of this extrapolation by measuring the apparent focal shift in samples where the refractive index is between 1.33 and 1.45 and with objectives with numerical apertures between 1.25 and 1.49.</p></div

Summary of a variety of methods used to measure relative focal shift.

<p>Values are reported for our highest NA objectives, NA 1.49, imaging into an aqueous environment, <i>n</i>₂ = 1.33.</p

Fluorescent images showing the disparity between slices in a single focal plane and along the focal dimension.

<p>Scale bars are 1 μm. (A-E) <i>E. coli</i> cell stained with a membrane dye (FM 4–64). (F-J) 1 <i>μ</i>m sphere with a fluorescent ring stain. (A,F) <i>xy</i> slice (B,G) <i>xz</i> slice showing the apparent elongation of the object along the focal axis. (C,H) Active contour fit to the ridge of maximal intensity in red. (D,I) Stretched circle fit in blue <i>r</i>² = (<i>αz</i>)² + <i>x</i>². (E,J) <i>xz</i> slice shown after scaling <i>z</i> by <i>α</i>.</p

Relative focal shift as measured as the distance between beads of different sizes.

<p>(A) Example images with a sample of three types of beads (100 nm TetraSpeck, 510 nm Dragon Green, 1040 nm Dragon Green) are shown at different sample positions. (B) Brenner gradients for three example beads highlighted in panel A. (C) Box and whisker plots of the full-width at half-maximum Brenner gradient (brown) and peak intensity (green). (D) Histograms of relative distances between beads and the plane of the smallest beads. (E) Sample motion needed to refocus from one size bead to another is proportional to the difference in their sizes. <i>α</i> is the slope of this line. Colors as in panel D. <i>x</i> error bars are standard deviations of observed positions. <i>y</i> error bars are 5% relative deviation in bead diameter. Solid line is the fit <i>y</i> = <i>αx</i> and the dashed lines are the 95% C.I. for <i>α</i>. (F) Standard deviation of bead positions, measured by Brenner gradient (brown) and peak intensity (green). 99% C.I. for <i>σ</i> is calculated from a bootstrap analysis and displayed as the error bars.</p

Properties of objectives used to test the versatility of the multiple bead method.

<p>Properties of objectives used to test the versatility of the multiple bead method.</p