Claims 23
From the paper MHz compression of pulse packets facilitates remote focusing of electroporation · claims file · checked 2026-10-22: ready
- fig7be-center-linear-with-bursts
In both CT26 and CHO cells, YP uptake at the array center increases linearly with the number of bursts (slopes different from zero at p < 0.001), except for saturation at 6 bursts (x4) in CT26 cells.
The increase was linear in both cell lines, except when it reached saturation after 6 bursts (x4) in CT26 cells
Results 3.4, paragraph 1; Figure 7B and 7E and legendHow it will be judged
Center YP is the background-subtracted intensity of the ROI at 0 mm, by the profile method, in a.u. A linear fit of center YP on burst number (1, 2, 4, 6 bursts (x4)), with the 6-burst point excluded in CT26 as the Fig. 7 legend says, and its slope tested against zero by a 2-sided one-sample t test (Fig. 7 legend). The paper does not say whether the fit is to individual experiments or to group means; the readings are (a) individual experiments and (b) group means. Burst number counted as 1, 2, 4, 6 or as 4, 8, 16, 24 total bursts gives the same p. Reproduced when, under one reading, both slopes are positive with p < 0.001; partially reproduced when that holds in one cell line only. A cell line absent from the dataset is reported as untested and the claim judged on the other.
- fig7b-ct26-saturation
In CT26 cells the central YP uptake saturates at 6 bursts (x4), falling below the linear increase seen from 1 to 4 bursts, while CHO cells show no saturation.
Considering that even 2 bursts (x4) caused strong cell death in the center ( Fig. 5 ), saturation was expected and indicated that all cells were already killed and YP staining reached its maximum
Results 3.4, paragraphs 1 and 2; Figure 7B and 7E legendHow it will be judged
The paper gives no test of saturation; it shows it by excluding the 6-burst point from the CT26 fit and by the 95% confidence band of that fit (Fig. 7 legend). Reproduced when the CT26 mean center YP at 6 bursts (x4) lies below the lower edge of the 95% confidence band of the linear fit through 1, 2 and 4 bursts, extrapolated to 6, and the CHO 6-burst mean lies within the band of the CHO fit through 1, 2 and 4 bursts. This band test is the file's reading; the paper's own CHO fit (Fig. 7E) uses all four burst numbers, and its significant slope is tested in fig7be-center-linear-with-bursts. Partially reproduced when the CT26 point lies below the fitted line but within the band. Readings (a) and (b) of fig7be-center-linear-with-bursts apply.
- fig7cf-fwhm-with-bursts
The FWHM of the central YP peak increases in proportion to the number of bursts in CT26 cells (slope p < 0.001) but not in CHO cells (p = 0.44).
Concurrently, FWHM of the central peak increased proportionally to the burst number in CT26 cells, but not in CHO cells
Results 3.4, paragraph 2; Figure 7C and 7F and legendHow it will be judged
FWHM from Lorentzian fits of each experiment's diagonal profile, by the profile method. CHO uses 2, 4 and 6 bursts (x4), since after one burst YP uptake was too small to measure FWHM (Fig. 7 legend); CT26 uses all four burst numbers (the legend excludes the 6-burst point from panel B only). Linear fit of FWHM on burst number with its slope tested by a 2-sided one-sample t test. Open details and their readings, the fit range (-4 to 4, -5 to 5, -6 to 6 mm), the ROI spacing (1 or 0.5 mm), and whether the linear fit is to individual experiments or to group means. Reproduced when, under one reading, the CT26 slope is positive with p < 0.001 and the CHO slope has p >= 0.05; partially reproduced when only one of the two holds.
- fig7-sharpest-peak-one-burst
One burst (x4) in CT26 cells gives the sharpest peak of the study, FWHM 1.1 +/- 0.1 mm, 7.3% of the inter-electrode gap and 2.3% of the electrode array area.
1 burst (x4) in CT26 cells produced the sharpest peak of electroporation seen in this study ... at FWHM of 1.1 ± 0.1 mm, it occupied just 7.3% of the inter-electrode gap and 2.3% of the electrode array area
Results 3.4, paragraph 2; Figure 7CHow it will be judged
Mean over CT26 experiments of the per-experiment Lorentzian FWHM at 1 burst (x4), by the profile method, with its tolerance the paper's stated spread (+/- 0.1 mm, a s.e.). The gap is the 15.3 mm diagonal (Results 3.3 gives 1.7 mm as 11.1%). The paper does not say how the area percentage is computed. Readings, (a) a disk of diameter FWHM over the square with 15.3 mm diagonals (117 mm2); (b) a disk of radius FWHM over the square of the electrode centers (12.8 mm sides, 164 mm2), the one reading found that gives 2.3% from 1.1 mm; (c) a disk of diameter FWHM over the square of the electrode centers. Reproduced when the FWHM and the gap percentage are within tolerance and the area percentage is within tolerance under one reading; partially reproduced when the FWHM alone is. Open details of the profile method (fit range, ROI spacing) are run under each reading.
- fig7-offtarget-40-50-fold
With 1 burst (x4) in CT26 cells, YP uptake near the electrodes is 40 to 50 times smaller than at the remote target in the center.
Off-target effects, characterized by YP emission near electrodes, were 40–50 times smaller than at the remote target
Results 3.4, paragraph 2How it will be judged
The reported range 40 to 50, widened by 10% of each bound, is 36 to 55, which the value and tolerance give. Background-subtracted YP by the profile method. Readings the text allows, near electrodes as (a) 6 mm or (b) 7 mm from the center (Results 3.1); the ratio as (i) of group means or (ii) the mean of per-experiment ratios. The group meant is also open. The sentence follows the 1 burst (x4) result, giving reading (A), 1 burst (x4) only; it closes a paragraph on all CT26 burst numbers, and a range of 40 to 50 can be the span over groups, giving reading (B), the ratios for 1, 2 and 4 bursts (x4) in CT26 (6 bursts saturates) each lie in 36 to 55. Reproduced when the ratio lies in 36 to 55 under one reading.
- fig7-cho-replicates-ct26
CANCAN remote focusing of electroporation found in CT26 cells also holds in CHO cells, which show a central peak of YP uptake that grows with burst number.
Increasing the number of bursts increased the amplitude of the central peak of YP uptake in both CT26 and CHO cells ( Fig 7A and D )
Results 3.4, paragraph 1; Conclusion; Figure 7DHow it will be judged
Diagonal profiles of CHO monolayers by the profile method. Reproduced when, for each burst number from 2 bursts (x4) up (after one burst CHO uptake was too small to measure FWHM, Fig. 7 legend; the 1-burst profile is reported), the mean center YP exceeds the mean YP at both 6 and 7 mm (the paper's measure of focusing, center against near electrodes, Results 3.1); together with the CHO part of fig7be-center-linear-with-bursts.
- fig4b-40-fold-at-02mhz
At a packet rate of 0.2 MHz, YP uptake in the center was 40-fold higher than near the electrodes, despite a roughly 4-fold stronger electric field near the electrodes.
Now, by increasing the packet repetition rate to 0.2 MHz, we achieved a 40-fold difference ( Fig. 4B ), despite using a bigger array with larger reduction of the electric field from electrodes to the center
Results 3.1, paragraph 3; Figure 4BHow it will be judged
Background-subtracted YP by the profile method, CT26, 2 bursts (x4), 0.2 MHz. Readings as for fig7-offtarget-40-50-fold, near electrodes at (a) 6 mm or (b) 7 mm, ratio (i) of group means or (ii) the mean of per-experiment ratios. Reproduced when the ratio is within tolerance under one reading.
- fig3a-cancan-center-enhanced
All CANCAN protocols enhance electroporation at the center of the array, while the unipolar control produces electroporation that decreases toward the center.
All CANCAN protocols visibly enhanced electroporation at the center of the array, in contrast to its gradual decrease toward the center in the control
Results 3.1, paragraph 2; Figure 3A to 3CHow it will be judged
CT26, 2 bursts (x4), diagonal profiles by the profile method. The paper's measure of focusing is the ratio of YP uptake in the center to that near electrodes (Results 3.1, paragraph 3). Reproduced when the group-mean ratio of center YP to YP at 6 mm and to YP at 7 mm exceeds 1 for every CANCAN packet rate and is below 1 for the control.
- fig3bc-offtarget-deviation-falls-with-rate
Increasing the packet rate from 1 Hz to 0.2 MHz suppresses electroporation near the electrodes and at the perimeter between electrodes, so that the excess of YP uptake over the Lorentzian fit at larger distances from the center diminishes with packet rate.
The deviation of experimental data from the fits at larger distances from the center diminished with increasing the packet repetition rate, highlighting better suppression of the off-target electroporation
Results 3.1, paragraphs 2 and 4; Figure 3B and 3CHow it will be judged
CT26, 2 bursts (x4), CANCAN packet rates, group-mean profiles along the diagonals (Fig. 3B) and along the lines through the center parallel to the sides (Fig. 3C), with the Lorentzian fitted from -4 to 4 mm as in fig3bc-lorentzian-profile. The paper gives no measure or test of the deviation. The measure used is the mean of observed minus fitted YP over the ROIs beyond 4 mm from the center, in % of the fitted peak a, which is the plain reading of "deviation of experimental data from the fits at larger distances". Reproduced when, in both directions, it is smaller at 0.2 MHz than at 1 Hz; the values for every rate are reported.
- fig3bc-lorentzian-profile
The decline of YP uptake sideways from the center is bell-shaped and follows a Lorentzian, with a nearly perfect fit within 4 mm of the center (R > 0.9, p < 0.001).
The fit correlation with data in the −4 to 4 mm radius was nearly perfect (R>0.9, p<0.001)
Results 3.1, paragraph 4; Figure 3B and 3C; Methods 2.5How it will be judged
CT26, 2 bursts (x4), CANCAN packet rates. Lorentzian fitted to the group-mean profile from -4 to 4 mm (the dotted fits of Fig. 3B and 3C), along the diagonals (Fig. 3B) and along the lines through the center parallel to the sides (Fig. 3C); the correlation R of fitted and observed values within that range. Reproduced when R > 0.9 with p < 0.001 for every rate and both directions; partially reproduced when it holds for most of them.
- fig4b-center-no-rate-effect
YP uptake in the center of the array shows no significant effect of the packet rate.
YP intensity in the center showed no significant effect of the packet rate ( t -test with Bonferroni correction, Fig. 4B )
Results 3.1, paragraph 5; Figure 4BHow it will be judged
A claim of absence, judged on the paper's test. Center YP (ROI at 0 mm) of CT26, 2 bursts (x4), at each packet rate compared with 1 Hz by two-sided Student's t-tests with Bonferroni correction over the comparisons, the same family as the Fig. 4C legend uses for the same experiments. Reproduced when no comparison has corrected p < 0.05; not reproduced when any does. Effect sizes are given either way; all pairs of rates are shown as a variant.
- fig4b-near-electrode-rate-decline
YP uptake at 6 and 7 mm from the center (near the electrodes) declines steeply with increasing packet rate, so that higher rates suppress off-target electroporation despite a stronger electric field there.
It contrasted with a steep, packet rate-dependent decline of YP uptake at 6 and 7 mm from the center (i.e., 1.65 and 0.65 mm from electrodes)
Results 3.1, paragraphs 2 and 5; Figure 4A and 4BHow it will be judged
CT26, 2 bursts (x4), YP at 6 and at 7 mm by the profile method. The paper fits a power function of packet rate (Fig. 4B legend) and gives no test. Reproduced when, at both distances, the power-function exponent fitted to YP against packet rate is negative and YP at 0.2 MHz is lower than at 1 Hz by a two-sided t-test at p < 0.05 (Methods 2.5). The paper does not say whether the power function is fitted to group means or to individual experiments; both are run.
- fig4c-fwhm-decreases-with-rate
Higher packet rates focus electroporation into a smaller spot, FWHM decreasing from about 4 mm at 1 Hz to about 2 mm at 1 kHz, 10 kHz and 0.2 MHz, less than 15% of the gap between electrodes.
FWHM decreased from ~4 mm at 1 Hz to ~2 mm at 1 kHz, 10 kHz, and 0.2 MHz packet rates (p = 0.03; 0.02, and 0.09, respectively; t -test with Bonferroni correction)
Results 3.1, paragraphs 6 and 7; Figure 4CHow it will be judged
CT26, 2 bursts (x4); per-experiment Lorentzian FWHM by the profile method, fitted from -4 to 4 mm (Results 3.1, Fig. 3 legend), averaged per rate. Each value is checked against its expected value. Each rate against 1 Hz by two-sided t-tests with Bonferroni correction (Fig. 4C legend). The paper's p for 0.2 MHz (0.09) is not below 0.05, so the claim's test is that the means fall within tolerance and the 1 kHz and 10 kHz comparisons reach corrected p < 0.05; the 0.2 MHz p is reported. Reproduced when all that holds and the smallest mean FWHM is below 15% of 15.3 mm; partially reproduced when the decrease is present but values miss tolerance or the tests fail.
- fig4c-log-dependence
FWHM decreases with the logarithm of packet rate from 1 Hz to 10 kHz (R = 0.77, p < 0.001), with no further gain in focality from 10 kHz to 0.2 MHz.
FWHM decrease followed the logarithmic dependence from 1 Hz to 10 kHz (correlation coefficient R = 0.77, p<0.001), but there was no further focality increase when the packet rate increased from 10 kHz to 0.2 MHz
Results 3.1, paragraph 6; Figure 4C legendHow it will be judged
Per-experiment FWHM as in fig4c-fwhm-decreases-with-rate; linear regression of FWHM on log of packet rate over the 15 experiments below 0.2 MHz (Fig. 4C legend), slope tested by a 2-sided one-sample t test. Reproduced when the slope is negative with p < 0.001 and |R| is within tolerance, and the 0.2 MHz mean FWHM is not lower than the 10 kHz mean by a two-sided t-test at p < 0.05 (Methods 2.5).
- fig5-raised-electrodes-weaker-center
With MHz-CANCAN there is practically no YP staining near the electrodes, and raising the electrodes 1 or 2 mm above the monolayer weakens electroporation in the central spot.
there was practically no YP staining near electrodes to start with, so the only clear effect of raising electrodes was the weakening of electroporation in the central spot
Results 3.2, paragraph 1; Figure 5A and 5BHow it will be judged
CT26, 0.2 MHz, 2 bursts (x4); center YP (ROI at 0 mm) by the profile method, in % of the 0 mm elevation peak (Fig. 5B legend). The paper gives no test. Reproduced when center YP at 1 mm and at 2 mm elevation is each lower than at 0 mm by a two-sided t-test with Bonferroni correction over the two comparisons at p < 0.05 (Methods 2.5); partially reproduced when only the 2 mm comparison holds. YP at 6 and 7 mm in % of the center is reported.
- fig5b-pr-gaussian-yp-lorentzian
The Pr (cell killing) profile is better fitted by a Gaussian than by a Lorentzian (R2 0.96 against 0.92), while the YP profile is better fitted by a Lorentzian (0.98 against 0.95).
For Pr data in the left panel of Fig. 5B , the coefficient of determination increased from 0.92 for Lorentzian fit to 0.96 for Gaussian fit. YP data showed the opposite trend, with the coefficients of 0.98 and 0.95 for Lorentzian and Gaussian fits, respectively
Results 3.2, paragraph 3; Figure 5BHow it will be judged
CT26, 0.2 MHz, 2 bursts (x4), electrodes at 0 mm elevation (the left panel of Fig. 5B is read as the 0 mm panel; the text does not name it). Group-mean diagonal profiles of YP and Pr in % of the peak, fitted from -5 to 5 mm (Results 3.2), with the Lorentzian and Gaussian of Methods 2.5. Since 10% tolerances on R2 near 1 are loose, the claim also needs the ordering. Reproduced when all four R2 are within tolerance, the Gaussian R2 exceeds the Lorentzian R2 for Pr and the Lorentzian exceeds the Gaussian for YP.
- fig5-yp-spot-slightly-larger-than-pr
The central spot of YP staining (electroporation) is just slightly larger than the spot of Pr staining (cell killing).
Somewhat unexpectedly, the central spot of YP staining (electroporation) was just slightly larger than the spot of Pr staining (cell killing; Fig 5A and B )
Results 3.2, paragraph 2; Figure 5A and 5BHow it will be judged
The spot size is read as the FWHM, the paper's measure of spot size (Results 3.1); YP FWHM from the Lorentzian and Pr FWHM as 2.4 sigma of the Gaussian (Methods 2.5), fitted to group-mean profiles from -5 to 5 mm at each elevation. Reproduced when the YP FWHM exceeds the Pr FWHM at 0 mm elevation. "Slightly" is not quantified in the paper and is not tested; the ratio is reported, and the 1 and 2 mm elevations as variants.
- fig5c-irreversible-center
The central spot shows bright YP staining of nuclei, indicating irreversible damage, while zones farther out show much weaker staining typical of reversible electroporation, and cells inside the electrode footprint are unstained though present.
a closer examination of the central spot confirmed bright YP staining of cell nuclei ( Fig. 5C , zone 1), which is indicative of the irreversible membrane damage and cell death. Reversible electroporation is distinguished by a much weaker YP staining
Results 3.2, paragraph 2; Figure 5CHow it will be judged
The paper judges nuclear staining by eye from high-resolution images and gives no measure of it, so that part is not tested. The testable part is the intensity. The zones are located only by rectangles in Fig. 5A (not seen); zone 1 is at the center, zone 3 reaches into an electrode footprint, zone 2 lies between. Reproduced when background-subtracted YP in a 325 x 406 um ROI at the center exceeds YP in ROIs at every distance from 2 mm to 7 mm along the diagonal (the paper places reversible electroporation from about 2 mm out, Results 3.2, paragraph 3), and Hoechst intensity inside the footprint is above background.
- fig6b-900ns-peak-not-significant
The central YP peak is higher for 900 ns pulses, but its difference from 300 and 600 ns pulses is not statistically significant.
The central peak of YP uptake was higher for 900-ns pulses, but the difference from the other two pulse durations was not statistically significant
Results 3.3, paragraph 2; Figure 6BHow it will be judged
A claim of absence. CT26, 0.2 MHz, 2 bursts (x4), center YP (ROI at 0 mm) by the profile method, n = 5 per duration. The paper does not name the test; Methods 2.5 names ANOVA and two-sided t-tests with Bonferroni correction, and Fig. 6D compares each duration with 300 ns. Tests run, (a) 900 ns against 300 ns and against 600 ns by two-sided t-tests with Bonferroni correction for two comparisons; (b) one-way ANOVA over the three durations. Reproduced when neither finds a difference at p < 0.05; not reproduced when both do; when they disagree, blocked as methods_underspecified with both shown. The 900 ns mean must be the highest for the first part of the statement; effect sizes are given.
- fig6c-normalized-decline-similar
Normalized to the central peak, the sideways decline of YP uptake looks similar for 300, 600 and 900 ns pulses.
When the peak YP emission was taken as 100%, its decline sideways was similar for the three tested pulse durations
Results 3.3, paragraph 2; Figure 6CHow it will be judged
A claim of absence of a difference, for which the paper gives no test. Profiles in % of each group's center value, by the profile method. Judged by the tests Methods 2.5 names, a one-way ANOVA over the three durations at each distance with Bonferroni correction over the distances. Reproduced when no distance shows a difference at corrected p < 0.05; not reproduced when any does, with the distances and effect sizes reported. The finer per-experiment FWHM analysis of fig6d-fwhm-sharper-longer-pulses is a separate claim.
- fig6d-fwhm-sharper-longer-pulses
Packets of 600 and 900 ns pulses give a significantly sharper central peak than 300 ns pulses; 900 ns pulses reduce the FWHM to 1.7 +/- 0.08 mm, 11.1 +/- 0.6% of the gap.
a more detailed analysis based on fitting of individual experiments (as described in 3.1 ) revealed significant sharpening of the central peak when packets were built from 600- and 900-ns pulses ( Fig. 6D ). The slope coefficient of the logarithmic fit of the data was different from zero at p = 0.003 (one sample, 2-sided t -test). 900-ns pulse packets reduced FWHM to 1.7 ± 0.08 mm, that was 11.1 ± 0.6% of the gap between electrodes
Results 3.3, paragraph 2; Figure 6D and legendHow it will be judged
CT26, 0.2 MHz, 2 bursts (x4), per-experiment Lorentzian FWHM by the profile method, tolerances the paper's stated spread (s.e.). Open details and their readings. The fit range is -5 to 5 mm (Fig. 6B legend) or -4 to 4 mm ("as described in 3.1"). The fit of FWHM on pulse duration is linear in the legend and logarithmic in the text; both are run. Each of 600 and 900 ns against 300 ns by two-sided t-tests with Bonferroni correction for two comparisons. Reproduced when, under one reading, both comparisons have corrected p < 0.05 with 600 and 900 ns sharper, the slope is negative with p < 0.05, and the 900 ns values are within tolerance; partially reproduced when the sharpening holds but the values miss tolerance or only one comparison is significant.
- fig2-heating-below-44c
Even in the worst-case heating protocol, temperature under the electrodes did not exceed 44 C, with heating above 30 C confined to about 2 mm from the electrodes after 2 bursts.
With 2 bursts ( Fig. 2A ), heating was restricted to an approximately 2-mm zone next to electrodes, where it could reach over 30 °C but did not exceed 44 °C
Methods 2.3, paragraph 2; Figure 2How it will be judged
Thermochromic film images of Fig. 2 after 2 and 6 bursts at 0.2 MHz without rotation. Reproduced when no part of the film shows the transition back to black (44 +/- 2 C) and, after 2 bursts, the blue color (30 +/- 2 C) occurs only within about 2 mm of an electrode.
- fig1e-field-simulation
In the simulation, 7.2 kV on one electrode produces 3.3 kV/cm in the center of the array and 9.4 and 13.5 kV/cm at 6 and 7 mm from the center toward the active electrode.
Specifically, 7.2 kV applied to one electrode produced the electric field of 3.3 kV/cm in the center, and 9.4 and 13.5 kV/cm at 6 and 7 mm from the center towards the active electrode, respectively
Methods 2.2, paragraph 4; Figure 1EHow it will be judged
These are outputs of the paper's Sim4Life Lite 7.0.1.8169 model (Methods 2.2), not measurements; the model files are not published, and the paper names no other source for them. Settled by the expected values if a public copy of the model is found; otherwise not testable from recordings.
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