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Additional Supporting Information may be found in the online version of this article.

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CYTO_22266_sm_SuppFig1.tif6132KSupporting Information Figure 1: Influence of optical section thickness on DNA distribution measurement in high-resolution confocal image-cytometry. DNA profiles (5000 events) have been obtained from confocal stacks that were acquired with different pinhole apertures to evaluate influences on DNA histogram parameters.
CYTO_22266_sm_SuppFig2.tif5200KSupporting Information Figure 2: Comparison of the bivariate distribution of DNA versus KI67 total fluorescence measured by FCM (left) and high-resolution (60x 1.35 NA oil-immersion objective) cytometry (right). Details of the sample preparation and acquisition for FCM were included as Supplementary Material.
CYTO_22266_sm_SuppFig3.tif14654KSupporting Information Figure 3: Intracellular KI67 regions can be analyzed as a statistically independent ensemble. KI67 positive regions detected intracellularly were plotted independently (129000 spots) from their cell of origin to measure their dimension (area x spot) and protein density (KI67 mean intensity per spot) (first row). Three different subpopulations were arbitrary set according to the spots-area distribution (inset), namely small (red dots, area <100 pixels (about 1 μm2), intermediate (green dots, area between 100 and 1000 pixels (about 10 μm2), and large KI67-spots (blue dots, area > 1000 pixels). DNA profiles (second row) and DNA- versus KI67- total fluorescence (third row) shows the distribution of the cells of origin containing at least one KI67-spot in the selected range.
CYTO_22266_sm_SuppFig4.tif43156KSupporting Information Figure 4: High-resolution Image cytometry provides different ways to identify mitotic cells. Isolation of cells according to high local protein density (DNA-content versus KI67 mean-signal per pixel per cell, A) complemented total protein expression profile and identify a subpopulation with defined physical properties (DNA content versus cell area, B) and high chromatin density (DNA mean signal per pixel per cell versus KI67 mean signal per pixel per cell, C) corresponding to the mitosis stage as demonstrated in the retrieved representative images (lower panel). Images were collected employing a widefield automated fluorescence microscope.
CYTO_22266_sm_SuppFig5.tif15274KSupporting Information Figure 5: Cell relocalization for high-resolution confocal cytometry. Using the ability of the A.M.I.CO software to read and write physical stage positions of the analysed cells, mitotic cells were first identified and gated after a first low resolution acquisition and then reacquired, using conditions that satisfy Nyquist requirements for spatial sampling (left panel). Representative 3D projections were reconstructed for selected positions to monitor KI67 distributions during the main mitotic stages (right panel).
CYTO_22266_sm_SuppInfo.doc24KSupporting Information

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