Speckle contrast fadeout is a measure of spindle microtubule turnover. (a) The ISI pulse induces clustering of the fluorescent molecules, increasing contrast. Any subsequent declustering process, such as diffusion and structural turnover, randomizes fluorophores’ positions, progressively driving contrast down to the FSM-like baseline. In the context of microtubule turnover, it is depolymerization (catastrophe) and polymerization of new microtubules that drives the system toward the low-contrast baseline. (b) A GFP–α-tubulin–tagged Drosophila S2 cell in metaphase before and ∼2 and ∼40 s after the ISI pulse (γ ≅ 2). Progressive contrast fadeout is observed because of microtubule turnover. The bottom row shows the same experiment after treatment with a low dose of Taxol (10 nM), which increases microtubule stability and, consequently, speckle persistence. Bar, 5 µm. (c) ISI-induced speckle fadeout is driven by the complementary effects of decreasing-intensity bright speckles and increasing-intensity dark speckles, a time window during which poleward speckle motion (flux) can be observed. (d) Contrast–time curves (lines) and their time point mean (dots) measured in rectangular areas enclosing the spindle in Drosophila S2 metaphase cells (n_control = 44, n_taxol = 31, $〈 γ 〉≅1.5−2.0$⁠). Microtubule half-lives for a double-exponential fitting are t_fast = 2.9 ± 0.4 s and t_slow = 21 × 2 s (R² = .999) for control and t_fast = 3.4 ± 0.8 s and t_slow = 17 ± 3 s (R² = .975) for Taxol-treated cells, which displays a baseline term y₀ = 0.38 ± 0.01 (see Materials and methods), indicating that a significant microtubule population (40%) persists for a time much longer than the duration of the experiment. This baseline compares to y₀ = 0.05 ± 0.01 in control cells.