Targeted gene expression

Our lab is interested in neural circuit function, and we use genetically encoded tools to describe the anatomy and patterns of activity in these circuits.  This requires us to express exogenous genes in targeted parts of the zebrafish nervous system. As a postdoc with Herwig Baier at UC-San Francisco, Ethan led a group of researchers in a Gal4 enhancer trap (ET) screen.  The screen generated nearly 200 lines of zebrafish expressing Gal4 in subsets of the nervous system, and these lines are an important part of how our lab targets proteins to circuits of interest.  The lines can be ordered from the Zebrafish International Resource Center, and images can be found at ZFIN.org.  Each line has a four digit identifier (Gal4s1000t, for example) that can be used in searching ZFIN.
 

Gal4 ET lines
A smattering of the lines from the San Francisco ET screen.
More details can be found in:  Scott et al, 2007Scott et al, 2009Scott and Baier, 2009 and  ZFIN.org.


Since Gal4 can be used, in principle, to express any UAS-linked transgene, we have great flexibility with what we can express in the circuits from our Gal4 lines.  Examples include photoconvertible proteins, markers for subcellular compartments, optogenetic indicators of neural activity, and optogenetic proteins for activating or inactivating neurons.  Details of these anatomical and functional approaches are described on the lab’s Projects and Opportunities page.

Selective Plane Illumination Microscopy (SPIM)

SPIM is an approach in which a collimated laser beam is shaped into a thin plane of light, which is projected into a specimen.  Fluorescence then occurs in this plane, and the emitted light is imaged from an orthogonally-oriented imaging objective. Our design is customised for high-speed brain-wide calcium imaging in zebrafish, and incorporates open space around the larva to permit a range of sensory stimuli to be applied. 
 

SPIM diagram

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The SPIM principle - a plane of excitation light is formed in the specimen, and emissions are imaged at 90°. (Image: openspim.org, CC3.0)



Imaging components

Labelled illumination path

The image shows the illumination light path comprising laser launch, beam expander, a 50:50 splitter, and then two plane paths with a mechanical slit, a cylindrical lens, and an illumination objective. 

  • The 3D-printed imaging chamber (centre) has coverslip walls, and holds a fish larva in position to be illuminated by the planes.  A vertical imaging objective captures fluorescent emissions. 
  • Our imaging column (right) is as simple as possible: a 20x water-immersion lens, an emissions filter, a focusing lens, and a camera. 
  • The only additional components are two empty filter cubes.  These allow us to put light into the specimen through the imaging objective and to project holograms into the brain for optogenetics.  

 

A complete parts list for a simpler version of this microscope can be found in the supplemental information accompanying Thompson et al, 2016.  If you have any questions about parts or construction, feel free to contact Ethan.

Optical physics

The filter cubes on the imaging column allow us to apply light to the specimen through the imaging objective.  Spatial Light Modulators (SLMs) are devices that modulate the wavefront of coherent light resulting in a pattern of constructive and destructive interference.  With an SLM, therefore, we can target and sculpt light in the brain while performing calcium imaging.  By applying a Gerchberg-Saxton algorithm, we can produce arbitrary three-dimensional shapes within the brain, allowing for spatially controlled optogenetic manipulations.

sculpted beam focusing through agarose


A schematic (left) for how SLM-sculpted light is projected into the specimen, and an image (right) of sculpted light emerging from the objective.  These images are  adapted from Favre-Bulle et al, 2015, where we describe this technique in greater detail.