Claims 31
From the paper Sub-microsecond optical measurements of cell membrane charging and lesioning by pulsed electric fields · claims file · checked 2026-10-21: ready
- fig1-pole-dimming
With Di-8-ANEPPS and 1-us pulses, polarization grows with field strength up to 570 V/cm; at 780 V/cm the cathodic pole stops increasing and dims, and at 1000 to 1400 V/cm the poles show reduced or absent polarization while regions nearer the equator stay polarized and may polarize more than the poles.
At a higher electric field strength of 780 V/cm, polarization at the cathodic pole does not further increase but dims out, indicating a less efficient charging of the partially permeabilized membrane. Still stronger electric fields produce a distinct pattern of reduced or absent polarization at the poles ... while regions closer to the equator remain relatively intact and may polarize more than the poles
Figure 1 and its legend; Results 3.1, paragraph 1How it will be judged
Figure 1 shows images of one cell, which the paper does not identify; the test uses the Di-8-ANEPPS 1-us escalation cells (Fig. 4B, n = 5), collapsed profiles and pole values as in the common method. Reproduced when (1) the mean cathodic pole dF at 780 V/cm is not above that at 570 V/cm, and (2) at 1400 V/cm the largest mean dF of the cathode-facing hemisphere lies outside the +-5 degree polar arc. The paper gives no test or threshold for this; the two conditions are read from the legend's wording. Background readings (a) and (b) both run.
- fig2a-fluovolt-saturation
With FluoVolt, pole dF rose with field strength until it saturated at 66% (255 mV) at 360 V/cm at the cathodic pole and -83% (-320 mV) at 570 V/cm at the anodic pole; stronger fields did not increase the anodic response and decreased the cathodic one.
ΔF increased proportionally to the electric field strength until reaching apparent saturation at 66% ΔF (255 mV IMP) at 360 V/cm at the cathodic pole, and −83% ΔF (−320 mV IMP) at 570 V/cm at the anodic pole. Further escalation of the electric field strength did not increase the dye response at the anodic pole and even decreased it at the cathodic pole.
Figure 2A; Results 3.2, paragraph 2How it will be judged
Per-image pole dF (common method, Fig. 2 measure) over the 60 pulse images of each set, averaged per cell and then across the 4 cells. Reproduced when both values are within tolerance, the cathodic means at 570 and 780 V/cm are not above that at 360 V/cm by more than the tolerance, the anodic means at 780 to 1400 V/cm are not more negative than at 570 V/cm by more than the tolerance, and the cathodic means at 1000 to 1400 V/cm are below that at 360 V/cm.
- fig2a-fluovolt-stable
FluoVolt measurements were stable without pulses (no significant bleaching or photosensitization), and the response within each set stayed stable through the 60 pulses, even at 1000 V/cm and above.
Without PEF (first 40 datapoints, or ~ 7 s in each set), measurements were stable, indicating no significant photosensitization or dye bleaching ... but the dye response in each set stayed stable through the 60 pulses applied
Figure 2A; Results 3.2, paragraph 2How it will be judged
A claim of absence. The paper reports no test for it; the test it names for Fig. 2 is a 2-tailed paired t-test at p < 0.05 over cells (n = 4). Per set and pole, compare the start and the end of the 60 pulse images, and of the 40 baseline images. The paper does not say how many images form the start and the end; readings (a) the first and the last image, (b) the first 10 and the last 10 images. Reproduced when under both readings no set at 1000 to 1400 V/cm, and no baseline, shows a significant change; not reproduced when under both readings any does; blocked as methods_underspecified when the readings disagree. The size of each change is reported.
- fig2b-di8-climbs-to-570
Di-8-ANEPPS dF kept increasing with field strength up to 570 V/cm at both poles.
This dye has a lower sensitivity to membrane polarization (note the vertical scale different from Fig. 2A), but ΔF kept climbing up to the electric field strength of 570 V/cm at both cell poles.
Figure 2B; Results 3.2, paragraph 3How it will be judged
Per-image pole dF averaged over each set's 60 pulse images, per cell, then across the 5 cells. Reproduced when the mean |dF| at each pole increases at every step from 43 to 570 V/cm.
- fig2b-di8-bleaching
Di-8-ANEPPS responses were somewhat affected by bleaching, most visibly at 43 V/cm, where bleaching added about 5% to dF at the anodic pole and took about 5% from it at the cathodic pole.
The responses were somewhat affected by dye bleaching (particularly noticeable at 43 V/cm, where bleaching added ~5% to ΔF at the anodic pole and reduced it by the same ~5% at the cathodic pole.
Figure 2B; Results 3.2, paragraph 3How it will be judged
Per-image pole dF of the Fig. 2 measure (F0 the mean of the 40 baseline images), 43 V/cm set, mean across the 5 cells. The paper does not say how the shift was measured. As F0 is the baseline mean, a steady loss of F makes dF drift downward through the set at both poles, which matches the text's signs. Readings for the shift (a) per pole, the mean dF of the last 10 pulse images (91 to 100) minus that of the first 10 pulse images (41 to 50); (b) the offset common to both poles, the mean of the cathodic and the anodic set-mean dF over the 60 pulse images (the text says the shift is the same ~5% at both poles, and the field response is opposite in sign at the two poles). Reproduced when under one reading the shift (at each pole, for reading a) is within tolerance. The tolerance is the protocol's 10% of the reported value.
- fig2b-di8-decline-within-set
With Di-8-ANEPPS, responses to pulses of 780 V/cm and above decreased gradually within each set of 60 images.
In addition, responses to PEFs at and above 780 V/cm decreased gradually in each set of 60 images, consistent with expectations of adding membrane damage by additional electroporation pulses in each set
Figure 2B; Results 3.2, paragraph 3How it will be judged
The paper gives no test; it reads a direction. Per set, the mean |dF| at the start and at the end of the 60 pulse images, averaged across the 5 cells; start and end as in fig2a-fluovolt-stable, readings (a) first and last image, (b) first 10 and last 10. The Results do not name the pole; the Discussion, point (a), cites Fig. 2B for a progressive decrease at the cathodic pole, so the cathodic pole is tested and the anodic pole is shown as a variant. Reproduced when under both readings the cathodic end is below the start for every set from 780 to 1400 V/cm; blocked as methods_underspecified when the readings disagree. The 2-tailed paired t-test of Fig. 2 is reported for each set.
- fig2cd-stable-before-electroporation
Repeated alternating sets at 150 and 230 V/cm (480 pulses and 800 flashes) gave dF responses constant within and between sets.
Repeated sets of PEFs at 150 and 230 V/cm, delivered in alternation, produced ΔF responses that stayed constant both within each set and between the sets (Fig. 2C and D).
Figure 2C and 2D; Results 3.2, paragraph 4How it will be judged
A claim of absence, judged on the paper's test, the 2-tailed paired t-test at p < 0.05 of the Fig. 2 legend (n = 8). Per cell and set, the mean pole dF over the 60 pulse images (Fig. 2D); every set before the 1000 V/cm set is compared with the first set at the same field strength (the legend's dashed lines are the first-trial means), at each pole. Reproduced when none differs; not reproduced when any does. Within-set changes are shown as in fig2a-fluovolt-stable, as a variant.
- fig2c-1000-within-set
At 1000 V/cm the cathodic pole dF fell from 40 +- 10% at the start of the set to 26.6 +- 8% at its end, a trend that was not statistically significant.
In contrast, at an electroporating field strength of 1000 V/cm, ΔF at the cathodic pole decreased from 40 ± 10% at the beginning of the set to 26.6 ± 8% at the end of it. Although not statistically significant (p > 0.05)
Figure 2C; Results 3.2, paragraph 4How it will be judged
Tolerances are the paper's s.e. Per-image cathodic pole dF; start and end are not defined, readings (a) the first and the last image of the 60, (b) the first 10 and the last 10 images. Reproduced when under one reading both values are within tolerance and the 2-tailed paired t-test gives p > 0.05.
- fig2d-lasting-reduction
Set-averaged dF stayed nearly constant before the 1000 V/cm exposure but was about 20% lower in the sets after it.
ΔF values averaged across each set of 60 PEFs stayed nearly constant before the electroporation, but dropped by about 20% in the sets acquired after it (Fig. 2D, p < 0.05).
Figure 2D and legend; Results 3.2, paragraph 4How it will be judged
Per cell and set, the mean pole dF over the 60 pulse images; each set after the 1000 V/cm set compared by a 2-tailed paired t-test (n = 8) with the sets before it at the same field strength. The legend's dashed lines mark the first-trial means, and the text compares with the sets before electroporation, which stayed nearly constant; readings for the reference (e) the first set, (f) the mean of all sets before 1000 V/cm, at the same field strength. The text does not say which pole or how the 20% is pooled; readings (a) cathodic pole, (b) anodic pole, (c) each post-1000 set separately, (d) the mean over the post-1000 sets. Reproduced when under one reading the reduction is within tolerance and at least one post-1000 set is significantly lower at p < 0.05.
- fig3-dye-sensitivity
Fitted linearly through the origin over -150 to +150 mV IMPC, FluoVolt changes by 26% and Di-8-ANEPPS by 9.1% dF per 100 mV, with correlation coefficients 0.96 and 0.78.
The correlation coefficients of the fits equaled 0.96 for FluoVolt and 0.78 for Di-8 ANEPPS (due to a lower signal-to-noise ratio), with respective slope coefficients of 26% and 9.1% ΔF per 100 mV of IMP.
Figure 3 and legend; Results 3.3, paragraph 1How it will be judged
All 360 perimeter points of every cell in the 43 to 360 V/cm sets of 1-us pulses at 900 ns, dF against IMPC computed per cell and point (Eq. 1, tau from the fit of reference [20], an outside input). Points with IMPC from -150 to +150 mV fit by least squares through the origin (Grapher). Grapher's correlation coefficient for a fit through the origin is not defined in the paper; readings (a) Pearson r of dF with IMPC in that range, (b) the square root of the uncentred R2 of the fit through the origin. Radius readings (a) and (b) and background readings (a) and (b) of the header all run. Reproduced when the slopes are within tolerance and the correlation coefficients are within tolerance under one reading.
- fig3-fluovolt-nonlinear
FluoVolt departed from linearity from about +-200 mV IMPC, while Di-8-ANEPPS stayed nearly linear from -500 to +500 mV; so FluoVolt's early departure reflects its dynamic range, not electroporation.
The actual measurements with FluoVolt gradually became non-linear already at ±200 mV, while those for Di-8-ANEPPS stayed essentially linear throughout the tested range from −500 to +500 mV.
Figure 3 and legend; Results 3.3, paragraph 1How it will be judged
The paper judges this by eye from a LOESS smooth (Grapher, span not given) against the linear fit; it gives no threshold. Measure here, as the paper's figure compares them, the smoothed dF against the line through the origin of fig3-dye-sensitivity, over the same points. Readings for the smooth (a) LOESS with span 0.3, (b) 0.5, (c) 0.75, (d) means in 50-mV IMPC bins. A departure is the smooth lying closer to zero than the line by more than 10% of the line's value (the protocol's default tolerance). Reproduced when under every reading the FluoVolt smooth departs from the line at every |IMPC| from 220 mV (the paper's 200 mV plus 10%) to the end of the data on both sides, and the Di-8-ANEPPS smooth does not depart at any |IMPC| from 150 to 500 mV where the data have points (inside +-150 mV the line is fit to the same points, and near zero 10% of the line's value is smaller than the noise, so the test there would turn on noise alone).
- fig4a-fluovolt-limits
FluoVolt measured and calculated IMP matched only at 43 and 150 V/cm; with stronger 1-us pulses the pole dF peaked at 71 +- 10% (cathodic) and -83.5 +- 5% (anodic), about +260 and -330 mV.
However, this assumption held only for the applied fields of 43 and 150 V/cm, where measured and calculated IMP matched. With stronger PEFs, maximum ΔF reached 71 ± 10% and −83.5 ± 5% at the cathodic and anodic poles, respectively.
Figure 4A; Results 3.4, paragraph 1How it will be judged
Tolerances are the paper's s.e. Collapsed pole dF per cell and field strength, mean across the 4 cells; the maximum is the largest mean |dF| over field strengths at each pole. Measured IMP (dF / 26%) compared with the cell's pole IMPC by Student's t-test at p < 0.05 (Methods 2.5, which does not say paired or unpaired; readings (a) 2-tailed paired, (b) 2-tailed unpaired). The paper judges the match from Fig. 4A and reports no test for it. Reproduced when both maxima are within tolerance, the test finds no difference at 43 and 150 V/cm at either pole, and finds measured below calculated at either pole at each of 230 V/cm and above.
- fig4a-fluovolt-cathodic-decline
FluoVolt cathodic polarization fell from 71% at 360 V/cm to 45 to 50% at 1000 to 1400 V/cm.
Notably, polarization at the cathodic pole decreased from 71% at 360 V/cm to 45–50% at 1000–1400 V/cm, indicating that the more severe membrane damage prevented its polarization to the maximum.
Figure 4A; Results 3.4, paragraph 1How it will be judged
Collapsed cathodic pole dF, mean across the 4 cells, per field strength. The 360 V/cm tolerance is the paper's s.e. for the same 71% (Results 3.4, paragraph 1). Reproduced when the 360 V/cm value is within tolerance and each of the 1000, 1200 and 1400 V/cm values lies within 40.5 to 55%.
- fig4b-di8-limits
With Di-8-ANEPPS and 1-us pulses, measured IMP fell below the calculated value at 570 V/cm, where dF peaked at 57 +- 14% (cathodic) and -55 +- 4% (anodic), over 600 mV.
The measured IMP decreased below the calculated value when the electric field reached 570 V/cm, concurrently with reaching the maximum ΔF of 57 ± 14% at the cathodic pole and − 55 ± 4% at the anodic pole. These values corresponded to membrane polarization by over 600 mV, which is twice the value measured with FluoVolt.
Figure 4B; Results 3.4, paragraph 2How it will be judged
Tolerances are the paper's s.e. Collapsed pole dF per cell and field strength, mean across the 5 cells. Reproduced when the largest mean |dF| at each pole occurs at 570 V/cm and is within tolerance, and measured IMP (dF / 9.1%) is below the pole IMPC at 570 V/cm by Student's t-test at p < 0.05 (at either pole) but not below it at 43 to 360 V/cm. Methods 2.5 does not say paired or unpaired; readings (a) 2-tailed paired, (b) 2-tailed unpaired. The paper reports no test for this comparison.
- fig4b-first-10-pulses
Averaged over only the first 10 of the 60 pulses, Di-8-ANEPPS cathodic polarization reached 66 +- 15% (710 +- 200 mV), so the 600-mV maximum is not the dye's limit.
Polarization measured this way reached 66 ± 15% (710 ± 200 mV) at the cathodic pole, suggesting that the 600-mV maximum, averaged from 60 PEFs, was not the dye limit but the actual IMP
Figure 4B, dotted lines; Results 3.4, paragraph 3How it will be judged
Tolerance is the paper's s.e. As the common method, with the polarization image collapsed from the first 10 of the 60 pulse images; mean across the 5 cells, largest over field strengths. Reproduced when within tolerance and above the 60-image maximum of fig4b-di8-limits.
- fig4b-cathodic-asymmetry
Above 780 V/cm the pole IMP fell progressively with field strength, more at the cathodic pole; at 1400 V/cm the cathodic pole no longer charged and the anodic pole fell to about -30% dF (-330 mV).
As the electric field increased further, the IMP reached at the poles progressively decreased, and the reduction was more pronounced at the cathodic pole ... At 1400 V/cm, charging was no longer detectable at the cathodic pole (i.e., the membrane became fully permeable) and dropped to ~30% ΔF (~ −330 mV) at the anodic pole.
Figure 4B; Results 3.4, paragraph 4How it will be judged
Collapsed pole dF, mean across the 5 cells. Reproduced when (1) the mean |dF| at each pole decreases from 780 to 1000, 1200 and 1400 V/cm, (2) the fraction lost between 780 and 1400 V/cm is larger at the cathodic pole, (3) the cathodic pole dF at 1400 V/cm does not differ from zero by a one-sample t-test at p < 0.05 (the test of Methods 2.5), and (4) the anodic value is within tolerance.
- fig4c-equator-follows-cosine
Membrane regions nearer the equator were unaffected by pole electroporation and followed the cosine dependence of the Schwan equation up to 1000 V/cm.
Membrane areas closer to the equator ... appeared relatively unaffected and followed the cosine dependence predicted by Schwan formula up to 1000 V/cm (Fig. 4C).
Figure 4C and legend; Results 3.4, paragraph 4How it will be judged
A claim of absence of a deviation. The paper names no test and no angular range for "closer to the equator". Measured IMP (dF / 9.1%) and IMPC per perimeter point, averaged per cell within arcs about the two equator points; readings (a) +-15 degrees, (b) +-30 degrees from the equator, on each side of each pole hemisphere. At 570, 780 and 1000 V/cm, measured against calculated by Student's t-test at p < 0.05 (Methods 2.5, n = 5), which the paper does not say is paired; readings (c) 2-tailed paired, (d) 2-tailed unpaired. Reproduced when no arc differs at any of these strengths under (c) and (d); not reproduced when any differs under both; blocked as methods_underspecified when they disagree. The ratio of measured to calculated is reported.
- fig4d-short-pulses-same-limit
With 0.3-us pulses at higher fields (357 to 3320 V/cm), imaged at 275 ns, the maximum dF was 53 +- 4% (about 600 mV) at both poles, as with 1-us pulses, and the response changed with field strength in the same way.
Contrary to the expectations, the results were similar to those with 1-μs pulses in Fig. 4B: the maximum ΔF reached was 53 ± 4% (~600 mV) at both cell poles. The pattern of how the response changed with increasing PEF strength was not different from that with 1-μs PEFs either.
Figure 4D; Results 3.4, paragraph 5How it will be judged
Tolerance is the paper's s.e. Collapsed pole dF per cell and field strength, mean across the 10 cells. Reproduced when the largest mean |dF| at each pole is within tolerance, and, as with 1-us pulses (fig4b-cathodic-asymmetry), the mean |dF| at both poles declines at the field strengths above the one giving the maximum, more at the cathodic pole. The paper gives no test for "not different"; none is applied.
- fig5a-gd-extends-anodic
With 1 mM Gd3+ the anodic pole charged to -864 +- 44 mV, against -542 +- 57 mV in parallel controls (p < 0.001).
Indeed, we found that 1 mM Gd3+ extended the range of membrane charging at the anodic pole down to −864 ± 44 mV (p < 0.001 compared to −542 ± 57 mV in parallel control experiments, Fig. 5A).
Figure 5A; Results 3.5, paragraph 1How it will be judged
Tolerances are the paper's s.e. Collapsed anodic pole dF converted to IMP (dF / 9.1%), per cell and field strength (150 to 1400 V/cm). Which datapoints are compared is not stated; readings (a) the most negative group mean over field strengths in each group, compared at those strengths, (b) the two groups at the same field strength, at the strength giving the Gd3+ minimum, (c) each cell's own most negative value. Two-tailed unpaired t-test. Reproduced when under one reading both values are within tolerance and p < 0.001.
- fig5a-gd-linear
With Gd3+, the measured IMP stayed nearly identical to IMPC down to -780 mV, so the Di-8-ANEPPS calibration holds at high IMPs.
Furthermore, measured IMP remained nearly identical to the theoretical IMPC down to −780 mV, indicating that the dye response remained linear and its calibration was valid even at high IMPs.
Figure 5A; Results 3.5, paragraph 1How it will be judged
A claim of absence of a deviation. Gd3+ group, anodic pole, every field strength whose mean IMPC is -780 mV or less negative; measured IMP against IMPC by Student's t-test at p < 0.05 (Methods 2.5, n = 7), which the paper does not say is paired; readings (a) 2-tailed paired, (b) 2-tailed unpaired. Reproduced when none differs under (a) and (b); not reproduced when any differs under both; blocked as methods_underspecified when they disagree. The ratio of measured to calculated is reported.
- fig5a-gd-cathodic
At the cathodic pole Gd3+ cells reached 647 +- 44 mV, not significantly above controls, but Gd3+ prevented the decline of IMP with stronger fields.
At the cathodic pole, polarization in the presence of Gd3+ reached 647 ± 44 mV, a value not significantly higher than that in the control group without Gd3+. However, Gd3+ prevented the progressive decline in IMP as the electric field was increased.
Figure 5A; Results 3.5, paragraph 2How it will be judged
Tolerance is the paper's s.e. Collapsed cathodic pole IMP per cell and field strength. The comparison with controls is a claim of absence, judged on the 2-tailed t-test of the Fig. 5 legend at p < 0.05, Gd3+ (n = 7) against control (n = 5), with the readings for which datapoints of fig5a-gd-extends-anodic. "Prevented the progressive decline" has no test; read as the Gd3+ mean cathodic IMP at 1200 and 1400 V/cm being no lower than its maximum by more than the s.e., while the control mean declines from its maximum. Reproduced when the value is within tolerance, no reading finds Gd3+ significantly higher, and the decline condition holds.
- fig5a-gd-differences
Gd3+ and control data differ significantly for datapoints with IMPC below -700 mV and above 1200 mV.
Differences between Gd3+ and control group data are significant at p < 0.05 or better (2-tailed t-test) for datapoints below −700 mV and above 1200 mV.
Figure 5A legendHow it will be judged
Datapoints are field strengths, placed by their mean IMPC (the x axis of Fig. 5A, which the "mV" bounds refer to). For each field strength and pole, measured IMP of the Gd3+ against the control cells by a 2-tailed unpaired t-test. Reproduced when every datapoint with mean IMPC below -700 mV or above 1200 mV differs at p < 0.05.
- fig5b-conductivity-calibration
By Eq. (2), the effective membrane conductivity barely affects charging while it stays far below cytosol and medium conductivities; a 10-fold rise reduces IMP by about 2% and a 100-fold rise by 12%.
its 10-fold increase would go unnoticed, yielding just a ~2% IMP reduction, and a 100-fold increase would reduce IMP by just 12%.
Figure 5B; Results 3.5, paragraph 3How it will be judged
A computation from Eq. (2) with the constants of Methods 2.3 (lambda_i 0.4 S/m, lambda_o 1.64 S/m, R 8 um, d 5 nm, intact lambda_m 3e-7 S/m); it uses no recordings. The reduction is relative to IMPC, readings (a) 1 - f/1.5, (b) 1 - f(lambda_m)/f(3e-7 S/m). Reproduced when both values are within tolerance under one reading.
- fig5c-gd-reduces-conductivity
For most datapoints, Gd3+ reduced the electroporation-induced effective membrane conductivity 2 to 3-fold.
For most datapoints, Gd3+ reduced the electroporation- induced effective conductivity 2–3-fold.
Figure 5C; Results 3.5, paragraph 4How it will be judged
For each field strength and pole, the ratio of mean measured to mean calculated IMP in each group (Fig. 5C uses the datapoints of Fig. 5A, mean values), converted to effective conductivity through Eq. (2) as in fig5b-conductivity-calibration. Datapoints whose measured IMP is not below IMPC give no conductivity increase and are left out. Reproduced when more than half of the remaining datapoints have a control-to-Gd3+ ratio between 1.8 and 3.3.
- fig6a-ca-single-pulse
A single 1-us pulse caused clear Ca2+ entry at 1000 and 1200 V/cm, with the signal elevated through 5 min; the small rise at 780 V/cm was not significant.
Exposure to a single 1-μs pulse at escalating field strengths, from 0 to 1200 V/cm established a clear peak of Ca2+ entry at 1000 and 1200 V/ cm, and Ca2+ signal remained elevated through the 5-min observation period (Fig. 6A). A subtle ΔF increase after exposure at 780 V/cm was at the detection limit and not statistically significant.
Figure 6A; Results 3.6, paragraph 2How it will be judged
Fluo-4 dF (Methods 2.4), background-subtracted, F0 the mean before the pulse, 14 cells. The test is not named; the paper uses a one-sample t-test against zero for the pulse-train Ca2+ data (Results 3.6), applied here at p < 0.05. The window is not given; readings (a) the peak frame after the pulse, (b) the last 30 s of the recording. Reproduced when at 1000 and 1200 V/cm dF is significantly above zero under both windows, and at 780 V/cm it is not under reading (a) (a claim of absence, judged on that test).
- fig6b-ca-train-responders
After a train of sixty 1-us pulses at 6 Hz, 28% of cells responded strongly at 780 V/cm and nearly all at higher field strengths.
A strong Ca2+ response was recorded in 28% of cells exposed at 780 V/cm and in nearly all cells at higher intensities (Fig. 6B).
Figure 6B; Results 3.6, paragraph 3How it will be judged
Each cell exposed once; Fluo-4 dF as in Methods 2.4. The paper does not define a strong response. Readings (a) peak dF after the train above the mean plus 3 s.d. of the pre-train baseline of the same cell, (b) peak dF above 50%, (c) peak dF above 100%. Reproduced when, under one reading, the 780 V/cm fraction is within tolerance, at least 90% of cells respond at each higher strength, and the 650 V/cm fraction is 2 cells (5.9%).
- fig6b-ca-650
At 650 V/cm only 2 cells (5.9%) responded strongly, yet the mean Ca2+ signal at the end of recording differed from zero (p < 0.001) and was marginally higher than at 230 V/cm (p < 0.05).
At 650 V/cm, only 2 cells (5.9%) showed a strong response, but the mean Ca2+ signal by the end of recording was significantly different from zero (p < 0.001, one sample t-test) and marginally higher than after exposure at 230 V/cm (p < 0.05, single-tailed t-test).
Figure 6B; Results 3.6, paragraph 3How it will be judged
Fluo-4 dF per cell (Methods 2.4). "By the end of recording" is not defined; readings (a) the last frame, (b) the mean of the last 10 s, (c) the mean of the last 30 s. One-sample t-test against zero at 650 V/cm; one-tailed unpaired Student's t-test of 650 against 230 V/cm. Reproduced when under one reading both p-values are within their bounds.
- fig7b-charging-during-pulse
Normalized to 150 V/cm, pole polarization during the conditioning pulse fell 1.5 times below linear scaling at 560 V/cm and 2-fold at 760 V/cm, and further at stronger fields (p < 0.01 for every point from 560 V/cm).
Actual measurements fell 1.5 times below this line for 560 V/cm PEFs, 2- fold for 760 V/cm PEFs, and diverged further for more intense PEFs (p < 0.01 for all datapoints ≥560 V/cm, one sample t-test).
Figure 7B, top panels; Results 3.7, paragraph 5How it will be judged
Data are those of Fig. 4B (Di-8-ANEPPS, 1-us pulses, no test pulse), pole dF as in fig4b-di8-limits, each strength as % of the 150 V/cm value, compared with the linear line (100% x E / 150 V/cm). Not stated whether normalization is per cell or of the means; readings (a) per cell, (b) of the means (the ratios), with the test per cell under both. Which pole is not stated; readings (c) each pole, (d) the mean of the poles. One-sample t-test of the per-cell values against the line. Reproduced when under one reading both ratios are within tolerance, the ratio at 1000 to 1400 V/cm exceeds 2, and every datapoint from 560 V/cm is below the line at p < 0.01.
- fig7ab-test-pulse-recovery
A 150 V/cm test pulse 5 us after the conditioning pulse charged the poles as in undamaged cells after conditioning up to 760 V/cm, to about 80% (not significant) after 1000 V/cm, and to about 70% and 50% after 1200 and 1400 V/cm, a persistent permeabilization.
Polarization still followed a cosine function after conditioning PEFs of up to 1000 V/cm. However, 1200 and 1400 V/cm conditioning PEFs markedly hindered charging by the test pulse at cell poles, indicating a persistent membrane permeabilization ... In contrast, measurements 6 μs later (test pulse) did not differ from the control up to 760 V/cm strength of the conditioning PEF; they were at ~80% of the control reading after 1000 V/cm conditioning PEF but the difference was not statistically significant. Still more intense PEFs of 1200 and 1400 V/cm reduced charging by the test pulse to ~70% and ~ 50%, respectively (p < 0.05 and p < 0.01, two-sided t-test).
Figure 7A and 7B, bottom panels; Results 3.7, paragraphs 3 and 5How it will be judged
Collapsed pole dF at 900 ns into the test pulse, as % of the 150/150 V/cm value. The test, from the Fig. 7 legend, is a two-sided t-test of the difference from the undamaged membrane; readings (a) one-sample t-test of per-cell % values against 100%, (b) paired t-test of dF against the same cell's 150/150 value. Pole readings as in fig7b-charging-during-pulse. The 570/760 and 1000 V/cm points are claims of absence, judged on that test at p < 0.05. Reproduced when under one reading the three values are within tolerance, 1200 and 1400 V/cm meet their p bounds, and no point up to 1000 V/cm differs. The 1400 V/cm cathodic value that the paper drops as negative (Results 3.7, paragraph 6) is reported both ways.
- fig7c-fast-conductivity-drop
Within 6 us of the electroporating pulse, effective membrane conductivity at the poles fell by a factor of about 10 (range 7 to 20) for most datapoints; the cathodic pole, more conductive during the pulse, no longer differed from the anodic pole at the test pulse.
Fig. 7C shows a profound reduction in the λm,eff within just 6 μs after the electroporating pulse. For most datapoints, it dropped by a factor of 10 (range: 7–20). While the cathodic pole became more conductive than the anodic pole during the electroporating pulse ... no difference between the poles was detected by the test pulse.
Figure 7C; Results 3.7, paragraph 7How it will be judged
As Methods 2.3 and Results 3.7 give it. From the mean values of fig7b-charging-during-pulse and fig7ab-test-pulse-recovery, each pole and field strength, the measured fraction (of the linear line, and of the 150/150 control) converted to effective conductivity through Eq. (2) as in fig5b-conductivity-calibration. The 1400 V/cm cathodic test value is left out if negative, and a test value above 100% is set to 100%, as the paper did. Datapoints with no conductivity increase during the conditioning pulse are left out. A test value of 100% maps to the intact 3e-7 S/m (full resealing, fig7c-full-reseal-mild), which gives a finite ratio far above 22; the paper's "most datapoints" leaves open whether these count, readings (a) left out, (b) counted as outside the range. Reproduced when more than half of the counted datapoints fall within 6.3 to 22 fold, the cathodic conductivity exceeds the anodic during the conditioning pulse at 1000 V/cm and above, and at the test pulse the cathodic and anodic dF do not differ at any conditioning strength by a two-sided paired t-test at p < 0.05 over the 5 cells (the t-test of the Fig. 7 legend, applied between the poles; a claim of absence). The paper gives no test for the pole comparison; the ratio of the two poles' test-pulse conductivities is reported.
- fig7c-full-reseal-mild
After mildly electroporating 570 and 760 V/cm pulses the membrane resealed fully within 6 us; after stronger pulses a residual conductivity increase persisted.
For mildly electroporating PEFs of 570 and 760 V/cm, membrane resealed fully (within the accuracy of measurements) within just 6 μs. With more intense PEFs, the residual increased conductivity persisted for at least 6 μs, indicating the formation of more stable membrane lesions.
Figure 7C; Results 3.7, paragraph 7How it will be judged
Judged on the test of fig7ab-test-pulse-recovery. Reproduced when the test-pulse pole dF after 570 and 760 V/cm conditioning does not differ from the control (so the inferred effective conductivity is the intact value), after 1200 and 1400 V/cm it is significantly reduced at either pole, and after 1000 V/cm the mean is below 100% at both poles (the statement's "more intense PEFs" include 1000 V/cm, whose ~80% is not significant; Fig. 7C is computed from the mean values, Fig. 7 legend).
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