Distribution and photostability of Fab310 conjugated with different fluorescent dyes in living cells.

<p>(<b>A</b> and <b>B</b>) Enrichment of dye-labeled Fab310 in the nucleus in living cells. HeLa cells were loaded with dye-labeled Fab310 and single focal planes were collected 4–6 h after loading. Fluorescence and phase-contrast images of a typical cell loaded with Alexa488-labeled Fab310 are shown in (<b>A</b>). Bar 10 µm. The enrichment of Fab310 in the nucleus was quantified by measuring the fluorescence intensities in the nucleus (yellow dotted area) and surrounding cytoplasm (4.8 µm-width area outside the nucleus, between yellow and green dots). The nucleus∶cytoplasm ratios of dye-labeled Fab310 (averages with s.d.; n = 6–7) are plotted in (<b>B</b>). The dyes showing higher nucleus∶cytoplasm ratios are suitable for live imaging. (<b>C</b>–<b>E</b>) Photostability of dyes conjugated with Fab310. HeLa cells loaded with dye-labeled Fab310 were continually exposed to excitation light for 10 min, during which images were captured every 2 sec. (<b>C</b>) Example image series for Alexa488- and Cy3-labled Fab310. The intensity of Alexa488 became apparently dimmer while that of Cy3 remained constant. Bar 10 µm. (<b>D</b>) Changes in fluorescence intensity by continuous excitation. Fluorescence intensities in nuclei were measured and plotted as relative values to the first image. Five different cells were analyzed for each dye-labeled Fab310. (<b>E</b>) Summary of photostability. The curves shown in (<b>D</b>) were fit to exponential decay curves. The bleach half time (t_1/2) and relative intensity after 10 min exposure are shown with s.d. (n = 5). Due to little bleaching under the condition used in the study, the t_1/2 could not be determined for Chromeo546, Cy3, and Dy548 (ND; not determined). The dyes showing less bleaching are suitable for live imaging.