Claims 30
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-pattern-poles-leaky
With Di-8-ANEPPS and 1 us PEFs, polarization depolarizes the cathode-facing pole and hyperpolarizes the anode-facing pole, falls off towards the equator and scales with field strength up to 570 V/cm; at 780 V/cm the cathodic pole stops increasing and dims; at 1000 to 1400 V/cm the poles show reduced or absent polarization while regions nearer the equator remain polarized and may exceed 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
Results, section 3.1, paragraph 1; Figure 1 and legendHow it will be judged
Figure 1 shows representative images; the paper does not identify the cells. Tested on the Di-8-ANEPPS 1 us escalation data (the Fig. 4B group, n = 5), with polarization images as in section 2.3 and perimeter dF resampled to 360 points. Reproduced when, in the mean over cells, (1) cathodic pole dF is positive and anodic pole dF negative at 43 to 570 V/cm and pole |dF| rises with field strength over that range; (2) cathodic pole dF at 780 V/cm is not above that at 570 V/cm; (3) at 1000 to 1400 V/cm pole |dF| is below its value at 570 V/cm at the cathodic pole, while the largest |dF| on the cathodic half of the profile lies away from the +-5 degree pole arc in at least one of those fields. Partially reproduced when (1) holds and one of (2) or (3) fails.
- fig2a-fluovolt-saturation
In FluoVolt-loaded cells, pole dF rose with field strength until apparent saturation 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.
Results, section 3.2, paragraph 2; Figure 2AHow it will be judged
Fig. 2A, n = 4 cells, 9 sets escalating from 43 to 1400 V/cm. Pole dF per image against F0, the mean of the set's first 40 images (section 2.3), averaged over the 60 PEF images of the set and then over cells. Reproduced when both values are within tolerance, the cathodic maximum over fields is at 360 V/cm or within its tolerance of the 360 V/cm value, the anodic dF at fields above 570 V/cm is not more negative than at 570 V/cm by more than the tolerance, and the cathodic dF at the strongest field is below its 360 V/cm value.
- fig2a-fluovolt-baseline-stable
Without PEF (the first 40 images of each set) FluoVolt measurements were stable, showing no significant photosensitization or dye bleaching.
Without PEF (first 40 datapoints, or ~ 7 s in each set), measurements were stable, indicating no significant photosensitization or dye bleaching.
Results, section 3.2, paragraph 2; Figure 2AHow it will be judged
A claim of absence for which the paper names no test. Methods 2.5 names linear regression with fit significance (Grapher) and paired t-tests (Fig. 2 legend), so two readings are run, per pole and set, on the 40 baseline dF values averaged over the n = 4 cells. (a) linear regression of dF on image index, fit significance p < 0.05; (b) 2-tailed paired t-test across cells of the mean of baseline images 1 to 20 against 21 to 40, p < 0.05. Reproduced when neither finds a significant change in any set at either pole, not reproduced when both do in the same sets; when the readings disagree the claim is blocked as methods_underspecified. The slope and the change in dF over the 40 images are reported.
- fig2a-fluovolt-stable-electroporating
At 1000 V/cm and above, which strongly electroporated the cells, the FluoVolt response stayed stable through the 60 pulses of each set.
Field strengths of 1000 V/cm and higher caused strong electroporation (see Section 3.5), but the dye response in each set stayed stable through the 60 pulses applied
Results, section 3.2, paragraph 2; Figure 2AHow it will be judged
The paper names no test. Readings as in fig2a-fluovolt-baseline-stable, run on the 60 PEF images of the sets at 1000 V/cm and above, per pole; (b) compares PEF images 1 to 30 with 31 to 60. Reproduced when neither reading finds a significant change; not reproduced when both do; blocked as methods_underspecified when they disagree.
- fig2b-di8-climbs-to-570
Di-8-ANEPPS has a lower sensitivity than FluoVolt, but its dF kept rising 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.
Results, section 3.2, paragraph 3; Figure 2BHow it will be judged
Fig. 2B, n = 5 cells; pole dF per set as in fig2a-fluovolt-saturation. Reproduced when the cathodic dF increases and the anodic dF becomes more negative at each step from 43 to 570 V/cm in the mean over cells, and the largest pole |dF| up to 570 V/cm is smaller than the FluoVolt value at the same field and pole (Fig. 2A group).
- fig2b-di8-bleaching-43
Di-8-ANEPPS responses were somewhat affected by bleaching, most noticeably at 43 V/cm, where bleaching added about 5% to dF at the anodic pole and reduced it by about 5% 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.
Results, section 3.2, paragraph 3; Figure 2BHow it will be judged
The paper does not say how the bleaching contribution was measured. Readings allowed by the text (a) the drift of pole dF over the 100 images of the 43 V/cm set, from a linear regression of the 40 baseline values extrapolated over the 60 PEF images (the drift adds to the hyperpolarizing anodic response and subtracts from the depolarizing cathodic one); (b) the difference in dF between the first and the last 10 images of the 43 V/cm set. Reproduced when, under a reading, both poles show a downward drift of F whose size is within tolerance; partially reproduced when the direction holds at both poles but the size is outside the tolerance under every reading.
- fig2b-di8-decline-within-set
Di-8-ANEPPS responses to PEFs at and above 780 V/cm 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
Results, section 3.2, paragraph 3; Figure 2BHow it will be judged
Fig. 2B, n = 5 cells. The paper names no test. Readings, per set at 780 V/cm and above, on pole |dF| averaged over cells (a) linear regression of |dF| on PEF image index, negative slope with fit significance p < 0.05; (b) 2-tailed paired t-test across cells of PEF images 1 to 10 against 51 to 60 (the first 10 of 60 are the window used in Fig. 4B), lower at the end with p < 0.05. The paper does not name the pole; readings (i) the cathodic pole, (ii) the anodic pole, (iii) both. Reproduced when, under a reading of each kind, the response declines significantly within every set at 780 V/cm and above; partially reproduced when it does in some of them. The slope for every set and pole is reported, and the slope at 43 to 570 V/cm is shown for comparison.
- fig2cd-nonelectroporating-stable
Repeated alternating sets of 150 and 230 V/cm PEFs produced dF responses that stayed constant within each set and between sets, so that 800 flashes and 480 pulses had no obvious impact before the 1000 V/cm exposure.
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).
Results, section 3.2, paragraph 4; Figure 2C and DHow it will be judged
A claim of absence. Fig. 2C and D, n = 8 cells; set means of pole dF over the 60 PEF images. The paper's test, from the Fig. 2 legend, is a 2-tailed paired t-test of each set mean against the first set at the same field strength (the dashed lines mark the first trial's means), at p < 0.05. Reproduced when no 150 or 230 V/cm set before the 1000 V/cm exposure differs from the first set of its field strength at either pole; not reproduced when any does. The within-set change (first against last 10 PEF images) is reported as a variant, as are the differences in percent.
- fig2c-1000-within-set
At 1000 V/cm, 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)
Results, section 3.2, paragraph 4; Figure 2CHow it will be judged
Tolerances are the paper's s.e. The paper does not say how many images form the beginning and the end. Readings (a) the first and the last PEF image; (b) the mean of PEF images 1 to 10 and 51 to 60; (c) the mean of images 1 to 5 and 56 to 60. Reproduced when, under a reading, both means are within tolerance, the end is lower, and a 2-tailed paired t-test across the 8 cells gives p > 0.05.
- fig2d-lasting-reduction
Set-averaged dF stayed nearly constant before electroporation but dropped by about 20% (a small but persistent reduction of at most 20%) in the 150 and 230 V/cm sets acquired after the 1000 V/cm exposure.
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).
Results, section 3.2, paragraph 4; Figure 2D and legendHow it will be judged
Fig. 2D, n = 8 cells. Set means of pole dF over the 60 PEF images, each set compared with the first set of the same field strength (the legend's dashed lines) by a 2-tailed paired t-test across cells. The paper does not say which pole or how the 20% is summarized. Readings (a) each pole separately; (b) both poles' |dF| averaged; and, for the size, (i) the mean reduction over all post-1000 V/cm sets, (ii) the reduction of each set. Reproduced when, under a reading, the post-exposure sets show a reduction within tolerance with p < 0.05 for at least one set, and no pre-exposure set differs at p < 0.05. Partially reproduced when the reduction is significant but its size is outside the tolerance under every reading.
- fig3-dye-slopes
Fitting dF against IMPC from -150 to +150 mV with a line through the origin gave slopes of 26% (FluoVolt) and 9.1% (Di-8-ANEPPS) dF per 100 mV, with correlation coefficients of 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.
Results, section 3.3, paragraph 1; Figure 3 legendHow it will be judged
From section 3.3 and the Fig. 3 legend. Cells exposed to 1 us PEFs at 43 to 360 V/cm, imaged at 900 ns; every perimeter location's dF (polarization against baseline image, 360 points per cell) paired with IMPC from Eq. (1) for that location, the cell's radius and its tau from the fit of [20] (outside input). All points from -150 to +150 mV IMPC, pooled over cells and fields, fit by least squares with a line through the origin; the correlation coefficient of that fit. The paper does not say which cells form Fig. 3; readings (a) the Fig. 4A and 4B groups at 43 to 360 V/cm; (b) every cell of the dye at those fields in the dataset. It does not say whether the correlation coefficient is for the through-origin fit or an ordinary Pearson r; both are shown. Reproduced when, under a reading, both slopes are within tolerance; partially reproduced when one is. The coefficients are reported against their values.
- fig3-fluovolt-nonlinear
FluoVolt measurements became non-linear already at +-200 mV, while Di-8-ANEPPS stayed essentially linear from -500 to +500 mV, so FluoVolt's early departure reflects the limit of the dye's range and 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.
Results, section 3.3, paragraph 1; Figure 3 legendHow it will be judged
Data as in fig3-dye-slopes over the whole IMPC range. The paper judged linearity by eye against a LOESS curve and names no test, so the method is open. Readings, using the tests Methods 2.5 names (a) per 50 mV bin of IMPC (a bin width not given by the paper), the per-cell mean dF compared with the through-origin line by a one-sample t-test across cells at p < 0.05; (b) a regression of dF on IMPC with a cubic term added to the through-origin line, over the whole range, with the fit significance of that term at p < 0.05. Reproduced when, under both readings, FluoVolt departs from the line beyond +-200 mV (bins beyond 200 mV on at least one side significantly below the line in magnitude, or a significant cubic term shrinking the response) and Di-8-ANEPPS does not depart within -500 to +500 mV; not reproduced when both readings find the opposite for either dye; blocked as methods_underspecified when the readings disagree. The per-bin ratios of measured to line are reported.
- fig4a-fluovolt-limits
FluoVolt-measured and calculated IMP matched only at 43 and 150 V/cm; with stronger PEFs pole dF reached at most 71 +- 10% (cathodic) and -83.5 +- 5% (anodic), about +260 and -330 mV, and the cathodic value fell from 71% at 360 V/cm to 45 to 50% at 1000 to 1400 V/cm.
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. ... Notably, polarization at the cathodic pole decreased from 71% at 360 V/cm to 45–50% at 1000–1400 V/cm
Results, section 3.4, paragraph 1; Figure 4AHow it will be judged
Fig. 4A, n = 4 cells, 1 us, 43 to 1400 V/cm; pole dF from the polarization image, mean over cells; IMPC at the poles from Eq. (1) with tau from [20]. Tolerances for the maxima are the paper's s.e.; the 45 to 50% range is widened by 10% of each bound (40.5 to 55). Reproduced when both maxima are within tolerance, cathodic dF at each of 1000, 1200 and 1400 V/cm (those present) lies within 40.5 to 55%, and measured IMP (dF / 0.26 per mV) is within the s.e. of IMPC at 43 and 150 V/cm but below IMPC by more than its s.e. at 230 V/cm and above at both poles (the text says the match held only at 43 and 150 V/cm).
- fig4b-di8-threshold-570
With Di-8-ANEPPS, measured IMP fell below the calculated value when the field reached 570 V/cm, together with the maximum dF of 57 +- 14% at the cathodic pole and -55 +- 4% at the anodic pole, a polarization of over 600 mV, twice that measured with FluoVolt.
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
Results, section 3.4, paragraph 2; Figure 4BHow it will be judged
Fig. 4B, n = 5 cells; pole dF from the polarization image (60 PEF images), mean over cells; IMP = dF / 0.091 per mV; IMPC from Eq. (1) with tau from [20]. Tolerances are the paper's s.e. Reproduced when both maxima are within tolerance and occur at 570 V/cm, and measured IMP at the poles is within the s.e. of IMPC at the fields below 570 V/cm and below it at 570 V/cm.
- fig4b-first10-cathodic
Averaging dF over the first 10 of the 60 PEFs, cathodic pole polarization reached 66 +- 15% (710 +- 200 mV), above the 600 mV maximum averaged over 60 PEFs.
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
Results, section 3.4, paragraph 3; Figure 4B (dotted lines)How it will be judged
Fig. 4B, n = 5 cells; the polarization image formed from the first 10 of the 60 PEF images (Fig. 4B legend), baseline image from the 40 baseline images. Tolerance is the paper's s.e. Reproduced when the maximum over fields of the mean cathodic dF is within tolerance and above the maximum from all 60 PEF images.
- fig4b-asymmetric-decline
Above 780 V/cm the IMP reached at the poles decreased progressively with field strength, more at the cathodic pole; at 1400 V/cm charging was no longer detectable at the cathodic pole and dropped to about 30% dF (about -330 mV) at the anodic pole.
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.
Results, section 3.4, paragraph 4; Figure 4BHow it will be judged
Fig. 4B, n = 5 cells, pole dF from the polarization image. "No longer detectable" is read as a mean cathodic dF at 1400 V/cm whose s.e. interval includes 0. Reproduced when pole |dF| decreases from 780 V/cm to 1400 V/cm at both poles, the fractional decrease from the pole's maximum is larger at the cathodic pole, the cathodic value at 1400 V/cm is not distinguishable from 0, and the anodic value is within tolerance. Note that -330 mV at 9.1% per 100 mV is -30% dF; the text prints the dF without its sign.
- fig4c-equator-cosine
Membrane regions nearer the equator appeared unaffected and followed the cosine dependence predicted by the Schwan equation up to 1000 V/cm, even when the poles were electroporated.
Membrane areas closer to the equator (at polar angles closer to 0◦and 270◦) appeared relatively unaffected and followed the cosine dependence predicted by Schwan formula up to 1000 V/cm (Fig. 4C).
Results, section 3.4, paragraph 4; Figure 4CHow it will be judged
Fig. 4B data (n = 5), perimeter dF resampled to 360 points, IMP = dF / 0.091 per mV, against IMPC from Eq. (1). The paper does not say how far from the poles "closer to the equator" reaches. Readings (a) the points within 30 degrees of the equator; (b) within 45 degrees. The paper compares by eye; the measure is, separately for the cathodic and anodic halves, the mean measured IMP over the points in that range divided by the mean IMPC over the same points (a ratio of means, since IMPC tends to 0 at the equator), against 1 with the protocol's default tolerance of 10% (0.9 to 1.1). Reproduced when, under a reading, that ratio is within 0.9 to 1.1 at 780 and 1000 V/cm while the ratio in the +-5 degree pole arcs is below 0.9 at the same fields.
- fig4d-short-pulses-same-limit
With 0.3 us PEFs at 357 to 3320 V/cm (imaged at 275 ns), the maximum dF was 53 +- 4% (about 600 mV) at both poles, similar to 1 us pulses, and its pattern with increasing field strength did not differ from that with 1 us pulses.
Contrary to the expectations, the results were similar to those with 1-μs pulses ... 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.
Results, section 3.4, paragraph 5; Figure 4DHow it will be judged
Fig. 4D, n = 10 cells; pole dF from the polarization image, mean over cells. Tolerance is the paper's s.e. Reproduced when the maximum |dF| over fields is within tolerance at both poles, and, as in fig4b-asymmetric-decline, pole |dF| declines at fields beyond the maximum with the larger fractional decline at the cathodic pole. Partially reproduced when one pole's maximum is out of tolerance.
- fig5a-gd-anodic-extension
1 mM Gd3+ extended membrane charging at the anodic pole down 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).
Results, section 3.5, paragraph 1; Figure 5AHow it will be judged
Fig. 5A, Di-8-ANEPPS, 1 us, 150 to 1400 V/cm; anodic pole dF from the polarization image converted to IMP at 9.1% per 100 mV. Tolerances are the paper's s.e. The paper does not say how the extreme is taken. Readings (a) the most negative group mean over field strengths, with its s.e., the groups compared at that field by a 2-tailed two-sample t-test; (b) each cell's most negative IMP over field strengths, averaged per group and compared by a 2-tailed two-sample t-test. Reproduced when, under a reading, both values are within tolerance and p < 0.001; partially reproduced when the Gd3+ value is more negative with p < 0.05 but a value is out of tolerance.
- fig5a-gd-linear-to-780
With Gd3+, measured IMP stayed nearly identical to IMPC down to -780 mV, showing that the Di-8-ANEPPS response stayed linear and its calibration valid 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.
Results, section 3.5, paragraph 1; Figure 5AHow it will be judged
Fig. 5A Gd3+ group, n = 7; anodic pole IMP against IMPC per field strength, means and s.e. both ways, compared with the bisector of Fig. 5A. The paper names no test for "nearly identical". Readings (a) a one-sample t-test (Methods 2.5) of per-cell measured IMP minus IMPC against 0, not significant at p < 0.05; (b) the mean measured IMP within 10% of the mean IMPC (the protocol's default tolerance). Reproduced when, under both readings, every field strength whose mean IMPC is between 0 and -780 mV passes; not reproduced when both fail at any of them; blocked as methods_underspecified when they disagree.
- fig5a-gd-cathodic
At the cathodic pole, polarization with Gd3+ reached 647 +- 44 mV, not significantly higher than in controls, but Gd3+ prevented the progressive decline of IMP as field strength increased.
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.
Results, section 3.5, paragraph 2; Figure 5AHow it will be judged
Readings for the maximum and its comparison as in fig5a-gd-anodic-extension, at the cathodic pole. The absence of a difference is judged on the paper's test, a 2-tailed two-sample t-test at p < 0.05. Reproduced when, under a reading, the Gd3+ maximum is within tolerance and not significantly above the control's, and the Gd3+ cathodic IMP at the strongest field is not below its maximum by more than its s.e. while the control's is.
- fig5a-gd-significant-points
Differences between Gd3+ and control data are significant at p < 0.05 or better for the datapoints 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
Fig. 5A; per field strength and pole, measured IMP of the two groups compared by a 2-tailed two-sample t-test. The text does not say which axis the -700 and 1200 mV refer to. Readings (a) IMPC (horizontal axis) below -700 mV at the anodic pole and above 1200 mV at the cathodic pole; (b) measured IMP below -700 mV in the Gd3+ group for the anodic pole, with IMPC above 1200 mV for the cathodic pole. Reproduced when, under a reading, every datapoint in those ranges has p < 0.05.
- fig5b-eq2-sensitivity
By Eq. (2), effective membrane conductivity has little effect on charging while it stays much smaller than the cytosol and medium conductivities; a 10-fold increase gives about a 2% IMP reduction and a 100-fold increase about 12%.
As expected, effective conductivity had little impact on charging for as long as it remained much smaller than the cytosol and media conductivities ... its 10-fold increase would go unnoticed, yielding just a ~2% IMP reduction, and a 100-fold increase would reduce IMP by just 12%.
Results, section 3.5, paragraph 3; Figure 5BHow it will be judged
A computation from Eq. (2) with the parameters in section 2.3 (cytoplasm 0.4 S/m, medium 1.64 S/m, intact membrane 3e-7 S/m, R = 8 um, d = 5 nm); it uses no recordings. Reduction is 1 - f/1.5 (the ratio of calculated to measured IMP is 1.5/f) at 10 and 100 times the intact membrane conductivity. Reproduced when both are within tolerance. If Eq. (2) as printed (the extracted text garbles it) gives a value of f far from 1.5 at the intact conductivity, both its literal form and the form normalized so that the intact membrane gives 1.5 are shown.
- 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.
Results, section 3.5, paragraph 4; Figure 5CHow it will be judged
As the Fig. 5C legend says, per field strength and pole, the effective conductivity from the ratio of the group-mean measured IMP to the group-mean IMPC, interpolated on the Eq. (2) curve of fig5b-eq2-sensitivity. The range 2 to 3 widened by 10% of each bound is 1.8 to 3.3. Reproduced when the control-to-Gd3+ ratio lies in 1.8 to 3.3 for more than half of the datapoints where both groups show a reduction of IMP below IMPC; the ratios are listed.
- fig6a-single-pulse-ca
A single 1 us pulse at 0 to 1200 V/cm produced a clear Ca2+ entry at 1000 and 1200 V/cm, with the signal elevated through the 5 min observation; the small increase after 780 V/cm was at the detection limit and 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.
Results, section 3.6, paragraph 2; Figure 6AHow it will be judged
Fig. 6A; the legend says the means are across 14 cells, and does not say whether these are 14 cells per field strength or 14 cells in all; Fluo-4 dF against F0 the mean before the pulse, background-subtracted (section 2.4). The paper does not name the test or the time point. Readings for the test (a) one-sample t-test of dF against 0 (Methods 2.5); (b) two-sample t-test against the 0 V/cm group, both 2-tailed at p < 0.05; for the time, (i) the peak dF after the pulse, (ii) dF at the end of the 5 min recording. Reproduced when, under every reading, 1000 and 1200 V/cm give dF significantly above the comparison and 780 V/cm does not; not reproduced when every reading fails for 1000 or 1200 V/cm or finds 780 V/cm significant; blocked as methods_underspecified when the readings disagree. The size of the 780 V/cm effect is reported under each reading.
- fig6b-train-ca
With trains of 60 1 us pulses at 6 Hz (224 cells, each tested once), a strong Ca2+ response occurred in 28% of cells at 780 V/cm and nearly all cells at higher fields; at 650 V/cm only 2 cells (5.9%) responded strongly, but the mean signal at the end of recording was significantly above zero (p < 0.001, one-sample t-test) and marginally higher than at 230 V/cm (p < 0.05, single-tailed t-test).
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). 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).
Results, section 3.6, paragraph 3; Figure 6BHow it will be judged
Fig. 6B; Fluo-4 dF against F0 the mean before the train (first 30 s), background-subtracted. "By the end of recording" is read as (a) the last frame; (b) the mean of the last 10 s (5 frames at 2 s). The single-tailed test is a two-sample Student's t-test, 650 above 230 V/cm. The paper does not define a strong response, and no reading is given; the fractions (28%, 5.9%, nearly all at higher fields) are tested only if the dataset or a reader's answer gives the definition, and otherwise that part is blocked as methods_underspecified with the distribution of end-of-recording dF per field shown. Reproduced when the cell count is 224 with 28 to 54 cells per field, and under a reading of the end, both tests give p below their bounds; partially reproduced when one does.
- fig7a-test-pulse-cosine
Polarization by a 150 V/cm test pulse 5 us after a conditioning pulse still followed a cosine after conditioning PEFs up to 1000 V/cm, while 1200 and 1400 V/cm conditioning PEFs markedly hindered charging at the cell poles.
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.
Results, section 3.7, paragraph 3; Figure 7AHow it will be judged
Fig. 7A, n = 5; perimeter dF (360 points) during the test pulse, mean over cells, fit with a cosine of the polar angle by least squares (Grapher, Methods 2.5). The 150/150 V/cm cosine is the benchmark (legend). The paper judges "followed a cosine" by eye. Read here as the absence of flattening at the poles, the mean dF in each +-5 degree pole arc not below the profile's own cosine fit by more than its s.e. Reproduced when this holds at both poles for every conditioning strength up to 1000 V/cm, and for 1200 and 1400 V/cm the mean pole dF is below the benchmark cosine's pole value by more than its s.e. at both poles. The fit significance, r and residuals of every cosine fit are reported.
- fig7b-conditioning-sublinear
During the conditioning pulse, pole polarization normalized to 150 V/cm fell 1.5 times below linear scaling at 560 V/cm, 2-fold at 760 V/cm and further for stronger PEFs (p < 0.01 for all points at 560 V/cm and above, one-sample t-test).
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).
Results, section 3.7, paragraph 5; Figure 7B, top panelHow it will be judged
Section 3.7 says the top panel uses the Fig. 4B data (conditioning pulses without test pulses; n = 5), with pole dF at 150 V/cm taken as 100% and linear scaling as E/150 x 100%. The fields 560 and 760 V/cm are read as the 570 and 780 V/cm sets of Fig. 4B. The paper does not say whether normalization is per cell or of the group mean; readings (a) per cell, then averaged; (b) group mean over group mean. Per pole, the one-sample t-test of per-cell normalized values against the linear expectation. Reproduced when, under a reading, both ratios are within tolerance at both poles or in their mean, the ratio grows beyond 760 V/cm, and p < 0.01 at every field of 560 V/cm and above.
- fig7b-test-pulse-recovery
Polarization by the test pulse 6 us after the conditioning pulse did not differ from control up to 760 V/cm conditioning, was about 80% of control after 1000 V/cm (not significant), and was reduced to about 70% and 50% after 1200 and 1400 V/cm (p < 0.05 and p < 0.01).
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).
Results, section 3.7, paragraph 5; Figure 7B, bottom panelHow it will be judged
Fig. 7A data, n = 5; pole dF during the test pulse with the 150/150 V/cm value as 100%. Normalization readings as in fig7b-conditioning-sublinear. The legend calls the test a "two-way t-test for the difference from the undamaged membrane"; readings (a) a one-sample two-sided t-test of the normalized values against 100%; (b) a two-sample two-sided t-test against the 150/150 V/cm values. The paper does not say which pole; readings (i) each pole, (ii) the mean of both. Reproduced when, under a reading, the three values are within tolerance, 1200 and 1400 V/cm reach p < 0.05 and p < 0.01, and no conditioning strength up to 1000 V/cm reaches p < 0.05.
- fig7c-fast-resealing
Effective membrane conductivity at the poles dropped about 10-fold (range 7 to 20) within 6 us after the electroporating pulse; the cathodic pole was more conductive during the pulse but no pole difference was seen by the test pulse; after 570 and 760 V/cm the membrane resealed fully within 6 us, while stronger PEFs left residual conductivity.
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 ... 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. For mildly electroporating PEFs of 570 and 760 V/cm, membrane resealed fully (within the accuracy of measurements) within just 6 μs.
Results, section 3.7, paragraphs 6 and 7; Figure 7CHow it will be judged
As section 3.7 and the Fig. 7C legend say, effective conductivity per pole and field from the mean normalized values of Fig. 7B, interpolated on the Eq. (2) curve (fraction of IMPC is the normalized value over the linear expectation for the conditioning pulse, and the normalized value over 100% for the test pulse); values above 100% taken as 100%, and the negative cathodic value at 1400 V/cm omitted, as the paper did. The range 7 to 20 widened by 10% of each bound is 6.3 to 22. The paper does not say which datapoints "most" counts; readings (a) every pole and field shown in Fig. 7C; (b) only those whose test-pulse value is below 100% (residual conductivity). The paper names no test for the absence of a pole difference at the test pulse; it is read as a 2-tailed paired t-test (Methods 2.5) of the cathodic against the anodic normalized test-pulse value across the 5 cells, at each field, not significant at p < 0.05. Reproduced when, under a reading, the fold drop lies in 6.3 to 22 for most datapoints, the conditioning-pulse conductivity is higher at the cathodic pole at most fields, no field shows a pole difference at the test pulse, the test-pulse conductivities at 570 and 760 V/cm are at the intact value (fraction of control at or above 100% within s.e.), and those at 1200 and 1400 V/cm are above it.
Pipeline
- Paper
- done (1 of 1)
- Claims
- done
- Claims check
- done (ready)
- Attempt
- todo
- Questions
- none
- Verified
- none