Pipeline for obtaining mean 3D separation measurements with nm-scale accuracy.
Critical factors were: (1) prepare bright high signal to noise samples to reduce standard deviation (SD) of centroid determination; (2) use mounting media like ProLong Gold to suppress z-axis spherical aberration; (3) use 0.17 mm thick coverslips for all measurements including CA measurement; (4) align imaging system and image using only a small region within the center of camera to suppress SD of CA; and (5) use warmed PFA fixation without pre-extraction or detergent in fixation solution.
List of mean values of Delta, S(2D), S(3D), ML2D, ML3D between EGFP-Ndc80, Ndc80-EGFP, EGFP-Nuf2, Nuf2-EGFP, EGFP-Spc24, Spc24-EGFP, EGFP-Spc25, or Spc25-EGFP and Hec1-9G3. All values listed are mean ±S.D.
List of mean values of Delta, S(2D), S(3D), ML2D, ML3D, Z-offset, and zML3D (Z-offset subtracted) for core kinetochore proteins. The differences between ML3D and ML2D (ML3D-ML2D), ML3D and zML3D (ML3D-zML3D) are also listed. All values listed are mean ±S.D.
Estimation of kinetochore-kinetochore variability (Ksd) in our measurements.
The table of experimental or simulated results using fixed S value (60 nm), CDsd ((x, y, z) = (±4, ±4, ±8)), and CAsd ((x, y, z) = (±9.1, ±7.5, ±17.6)) with kinetochore-to-kinetochore variability (Ksd = ±0, ±5, ±10, or ±15 nm).
(A) List of Delta, S(2D), S(3D), Z-offset, and zS(3D) (Z-offset subtracted) for the mean separation between CENP-T(M) and Hec1(9G3) in samples with or without large Z-offset.
(B) List of Delta, S(2D), S(3D), Z-offset, and zS(3D) in samples with #1, #1.5, and #2 coverslips. The value zS(3D) was calculated from the S(3D) data after subtraction of the mean Z-offset in the middle of the spindle from the Sz values. All values were mean ±SD.
Computer simulations to determine measurement SD and to test the accuracy of using mean CA for CA correction.
(A) Comparison of ~60 nm experimental results to simulated results for a fixed S value of 60 nm, the estimated CDsd for our measurements, and with or without CAsd. (B) Simulated S(3D), and ML3D values for different input S values with fixed angles (0˚), CDsd (x, y, z) = (±4, ±4, ±8) and CAsd (x, y, z) = (±9.1, ±7.5, ±17.6). All simulated values were mean ±SD.