There are several commonly used approaches for the study of membrane properties of live cells based on fluorescence probes. In one approach, lipids with specific fluorescent markers are incorporated in the cell membranes. The advantage of this approach is that it is possible to study the membrane distribution of specific lipids. However, when the aim of the study is to detect membrane microdomains independently of their lipid composition, it is more useful to use a single probe that can report on the specific properties of the membrane microdomains, independently of the probe segregation in one specific domain and location in the cell. One fluorescent probe that has been successfully used for this purpose is the lipophilic probe Laurdan (2-dimethylamino-6-lauroylnaphthalene), originally synthesized by Weber and Farris.1 Different membrane environments produce marked changes both in the spectrum and in the fluorescence lifetime of Laurdan. The sensitivity of the emission spectrum of Laurdan to the environment originates from the specific molecular structure of Laurdan in which the excited-state dipole is substantially different from the ground-state dipole (Figure 13.1).

Chromatin organization and dynamics are critical for gene regulation. In this work we present a methodology for fast and parallel three-dimensional (3D) tracking of multiple chromosomal loci of choice over many thousands of frames on various timescales. We achieved this by developing and combining fluorogenic and replenishable nanobody arrays, engineered point spread functions, and light sheet illumination. The result is gentle live-cell 3D tracking with excellent spatiotemporal resolution throughout the mammalian cell nucleus. Correction for both sample drift and nuclear translation facilitated accurate long-term tracking of the chromatin dynamics. We demonstrate tracking both of fast dynamics (50 Hz) and over timescales extending to several hours, and we find both large heterogeneity between cells and apparent anisotropy in the dynamics in the axial direction. We further quantify the effect of inhibiting actin polymerization on the dynamics and find an overall increase in both the apparent diffusion coefficient D* and anomalous diffusion exponent α and a transition to more-isotropic dynamics in 3D after such treatment. We think that in the future our methodology will allow researchers to obtain a better fundamental understanding of chromatin dynamics and how it is altered during disease progression and after perturbations of cellular function.