Project Pipeline Support

PPS continues the delivery of FlyLight pipeline output (FlyLight Extension labeled brains and light microscopy images) on demand to Janelia labs after the FlyLight Project Team ramped down. PPS also supports the Fly-eFISH small Project Team, performing the EASI-FISH technique to label neuropeptides in the Drosophila brain and gut. 

Connectomics Annotation carries on from the Connectome Annotation Team (CAT) and Annotation & Analytics (A&A). We support the FlyEM Project Team and their collaboration with the Wellcome Trust, along with providing annotation services to Janelia labs. C&A generally operates at the synapse and whole neuron level, collaborating with Project Technical Resources (PTR) and the CellMap Project Team, which focuses more on intracellular structures in cells and tissues.

PPS works closely with PTR, sharing resources and coordinating support efforts as needed as they shrink and grow. PPS is geared to assist projects that are high-throughput and/or use established methods, and typically require a scheduling lead time of about 2 months. By contrast, PTR focuses on smaller projects that may be more developmental in nature, and strives to accommodate these on a more flexible time scale. PPS and PTR work closely together to find the best support solution for each project.

What We Do

Connectomics Annotation:

C&A continues our ongoing efforts in the Drosophila electron microscopy (EM) datasets towards connectomes, potentially expanding to additional data types acquired using light microscopy (LM), video, audio and other modalities.

We perform annotation using software tools and applications developed in-house, or available commercially or as open source. We have expertise in NeuTu, Neu3, and Neuroglancer for annotating neuron morphologies and connectomes with FlyEM, and deploy this expertise to aid Janelia labs with annotation strategies to meet specific goals.

We continue to develop our capabilities in new technologies. Using data science, visualization and other analysis approaches, we aim to assist Janelia scientists in converting data to specific knowledge.

FlyLight Extension:

FlyLight developed standard pipelines for visualizing genetic driver lines using GAL4, LexA, split-GAL4, and split-LexA. Visualizations include general neuron morphology, synaptic labeling to determine neuronal polarity, and single cell labeling (MCFO). Crosses are scheduled by pipeline with the Fly Facility, followed by CNS dissection, immunohistochemistry, mounting, and high-quality confocal imaging. We use Zeiss LSM 700, and 880 microscopes. Images can be delivered directly (as LSMs) or processed through alignment pipelines and organized in Janelia Workstation.

PPS continues to offer these services to Janelia labs at large or small scale. Our core work continues to be the high-throughput screening of Initial Split (IS) GAL4 combinations to label specific subsets of neurons. The Fly Facility then makes a stable stock with the same split halves, and we verify its expression pattern. The Stable Split (SS) stock can then be further analyzed in our Polarity or MCFO pipelines. SS lines are a lasting resource for other purposes such as EASI-FISH, single-cell sequencing, or electrophysiology.

EASI-FISH:

PPS is using EASI-FISH to compare neuropeptide and split-GAL4 expression in the fly for the Fly-eFISH Project Team. EASI-FISH (Expansion-Assisted Iterative Fluorescence In Situ Hybridization) identifies single RNA transcripts in expanded fly tissue with very low background and rapid light-sheet imaging. Multiplexing split-GAL4 lines and probes allows the comparison of tens of neuropeptides to each line. PTR continues to perform EASI-FISH as well, handling most custom requests. 

We encourage collaborations on a wider range of projects, such as dissecting other species, using alternate labeling methods, etc.