High throughput 3D serial section electron microscopy
Serial section electron microscopy can reveal all cell membranes and organelles in a piece of tissue. By automating the cutting, imaging, and analysis of serial section electron microscopy, it is possible to reconstruct every neuron and synapse in a local circuit. We use automated tape collection ultramicrotomy (ATUM) to cut thousands of serial sections and collect the sections onto a stable tape substrate. We then use scanning electron microscopy and custom acquisition software to map data sets at low resolution and then direct high-resolution imaging to volumes of interest.
Correlated light and electron microscopy (CLEM)
By combining 3DEM circuit reconstructions with optical imaging of the same neurons, it is possible to map molecular and physiological information onto reconstructions of the synaptic connectivity of neural circuits. Our approach to 3DEM imaging lends itself to CLEM because, prior to high-resolution imaging, we first generate a low-resolution millimeter-scale 3D map of all EM sections. This millimeter-scale 3D EM map can be readily correlated with prior optical imaging of the same tissue. Applications of this approach in the lab include optical targeting of specific cell types, functional characterization of neurons with calcium imaging, and targeting of specific retinal ganglion cell synaptic motifs for EM reconstruction.
3D optical imaging
The lab uses confocal imaging, two-photon imaging, and serial section widefield imaging to characterize cell types and synaptic connectivity in the retina and lateral geniculate nucleus.
We develop custom image analysis code in Matlab and python. Coding projects include automated cell segmentation of 3DEM volumes, management of multichannel optical image stacks, and navigation and visualization of dense circuit reconstructions.