We consider acquisition schemes that maximize the fraction of images that contain only a single activated molecule (as opposed to multiple activated molecules) in superresolution localization microscopy of fluorescent probes. During a superresolution localization microscopy experiment, irreversible photobleaching destroys fluorescent molecules, limiting the ability to monitor the dynamics of long-lived processes. Here we consider experiments controlled by a single wavelength, so that the bleaching and activation rates are coupled variables. We use variational techniques and kinetic models to demonstrate that this coupling of bleaching and activation leads to very different optimal control schemes, depending on the detailed kinetics of fluorophore activation and bleaching. Likewise, we show that the robustness of the acquisition scheme is strongly dependent on the detailed kinetics of activation and bleaching.

The use of semiconductor nanocrystals (quantum dots) as fluorescent labels for multiphoton microscopy enables multicolor imaging in demanding biological environments such as living tissue. We characterized water-soluble cadmium selenide-zinc sulfide quantum dots for multiphoton imaging in live animals. These fluorescent probes have two-photon action cross sections as high as 47,000 Goeppert-Mayer units, by far the largest of any label used in multiphoton microscopy. We visualized quantum dots dynamically through the skin of living mice, in capillaries hundreds of micrometers deep. We found no evidence of blinking (fluorescence intermittency) in solution on nanosecond to millisecond time scales.

A class of highly fluorescent and photostable core-shell nanoparticles from a modified Stober synthesis in the size range of 20-30 nm is described. These nanoparticles are monodisperse in solution, 20 times brighter, and more photostable than their constituent fluorophore, and are amenable to specific labeling of biological macromolecules for bioimaging experiments. The photophysical characteristics of the encapsulated fluorophores differ from their solution properties. This raises the possibility of tuning nanoparticle structure toward enhanced radiative properties, making them an attractive material platform for a diverse range of applications.

Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional activity within single Saccharomyces cerevisiae cells. We found that expression levels for particular genes are higher than initially reported and can vary substantially among cells. However, variability for most constitutively expressed genes is unexpectedly small. Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts. In contrast, PDR5, a gene regulated by the transcription coactivator complex SAGA, is expressed using transcription bursts, resulting in larger variation. These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome.