Biology subjects

Lavis, L. D.

Publications and source records attributed to Lavis, L. D..

39 records · Page 3Linked to original sources

bioRxiv2017

A general method to fine-tune fluorophores for live-cell and in vivo imaging

AO_SCPCAPBSTRACTC_SCPCAPPushing the frontier of fluorescence microscopy requires the design of enhanced fluorophores with finely tuned properties. We recently discovered that incorporation of four-membered azetidine rings into classic fluorophore structures elicits substantial increases in brightness and photostability, resulting in the Janelia Fluor (JF) series of dyes. Here, we refine and extend this strategy, showing that incorporation of 3-substituted azetidine groups allows rational tuning of the spectral and chemical properties with unprecedented precision. This strategy yields a palette of new fluorescent and fluorogenic labels with excitation ranging from blue to the far-red with utility in live cells, tissue, and animals.

cell biology↗

bioRxiv2017

Semisynthetic pH-Sensitive Fluorophores For Imaging Exocytosis And Endocytosis

The GFP-based superecliptic pHluorin (SEP) enables detection of exocytosis and endocytosis, but its performance has not been duplicated in red fluorescent protein scaffolds. Here we describe semisynthetic pH-sensitive protein conjugates that match the properties of SEP. Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis, constituting new bright sensors for these key steps of synaptic transmission.

cell biology↗

bioRxiv2017

Stochastic Protein Labeling Enables Long-term Single Molecule Observation In Vivo

Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of >10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a waterfall mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo.

biophysics↗