Scanned light sheet microscopy with confocal slit detection.

In light sheet fluorescence microscopy optical sectioning is achieved by illuminating the sample orthogonally to the detection pathway with a thin, focused sheet of light. However, light scattering within the sample often deteriorates the optical sectioning effect. Here, we demonstrate that contrast and degree of confocality can greatly be increased by combining scanned light sheet fluorescence excitation and confocal slit detection. A high frame rate was achieved by using the "rolling shutter" of a scientific CMOS camera as a slit detector. Synchronizing the "rolling shutter" with the scanned illumination beam results in confocal line detection. Acquiring image data with selective plane illumination minimizes photo-damage while simultaneously enhancing contrast, optical sectioning and signal-to-noise ratio. Thus the imaging principle presented here merges the benefits of scanned light sheet microscopy and line-scanning confocal imaging.

Activated STAT1 transcription factors conduct distinct saltatory movements in the cell nucleus.

The activation of STAT transcription factors is a critical determinant of their subcellular distribution and their ability to regulate gene expression. Yet, it is not known how activation affects the behavior of individual STAT molecules in the cytoplasm and nucleus. To investigate this issue, we injected fluorescently labeled STAT1 in living HeLa cells and traced them by single-molecule microscopy. We determined that STAT1 moved stochastically in the cytoplasm and nucleus with very short residence times (<0.03 s) before activation. Upon activation, STAT1 mobility in the cytoplasm decreased ∼2.5-fold, indicating reduced movement of STAT1/importinα/ß complexes to the nucleus. In the nucleus, activated STAT1 displayed a distinct saltatory mobility, with residence times of up to 5 s and intermittent diffusive motion. In this manner, activated STAT1 factors can occupy their putative chromatin target sites within ∼2 s. These results provide a better understanding of the timescales on which cellular signaling and regulated gene transcription operate at the single-molecule level.

Balbiani ring mRNPs diffuse through and bind to clusters of large intranuclear molecular structures.

A detailed conception of intranuclear messenger ribonucleoprotein particle (mRNP) dynamics is required for the understanding of mRNP processing and gene expression outcome. We used complementary state-of-the-art fluorescence techniques to quantify native mRNP mobility at the single particle level in living salivary gland cell nuclei. Molecular beacons and fluorescent oligonucleotides were used to specifically label BR2.1 mRNPs by an in vivo fluorescence in situ hybridization approach. We characterized two major mobility components of the BR2.1 mRNPs. These components with diffusion coefficients of 0.3 ± 0.02 µm²/s and 0.73 ± 0.03 µm²/s were observed independently of the staining method and measurement technique used. The mobility analysis of inert tracer molecules revealed that the gland cell nuclei contain large molecular nonchromatin structures, which hinder the mobility of large molecules and particles. The mRNPs are not only hindered by these mobility barriers, but in addition also interact presumably with these structures, what further reduces their mobility and effectively leads to the occurrence of the two diffusion coefficients. In addition, we provide evidence that the remarkably high mobility of the large, 50 nm-sized BR2.1 mRNPs was due to the absence of retarding chromatin.