Dr. Emma Sigfridsson
Centre for Clinical Brain Sciences, University of Edinburgh, UK
The group of Professor Seth Grant are interested in whole-brain synaptome mapping with an aim to understand more about how synaptic proteins are distributed and the role the molecular heterogeneity of synapses plays in health and brain disease.
There are more than 1000 genes encoding proteins in the excitatory postsynaptic density alone, and the group aims to catalog as many synaptic proteins and protein subtypes in both excitatory and inhibitory pre and post-synaptic densities.
The group mainly works with fixed mouse brain sections that contain genetically-encoded fluorescent synaptic proteins. So far, the group has categorized two proteins: PSD95 and SAP102, both found in excitatory postsynaptic densities. They have monitored the changes in the distribution of these proteins throughout different stages of development and aging in mice.
The group is also working to map the synaptome of models of disease as there is a link between mutations in the genes encoding proteins found in the postsynaptic density of excitatory synapses and neuropsychiatric and neurodevelopmental disorders in humans.
In addition, the group looks at human post-mortem tissue using immunohistochemistry to investigate synaptic proteins in the human brain and in the context of human neurodegenerative diseases such as Alzheimer’s.
The Grant group was previously using an Andor XD Revolution Spinning Disk system with an EMCCD camera. The main issue they faced was speed and throughput, partly due to the small imaging FOV and long exposure times needed for certain fluorophores.
The group found it necessary to image using a 100X objective in order to obtain maximum resolution. However, the brain slices are very large, around 16 mm x 8 mm in area, thus imaging at high magnification with limited FOV was very time-consuming. The FOV of the EMCCD is ~11 mm diagonal, however, the FOV is further restricted by using an additional 2x magnification changer to achieve Nyquist imaging with the large pixels of the EMCCD. With this small FOV, imaging whole coronal or sagittal sections of mouse brains can take more than 10 hours to image just two channels.
The group also found signal levels of certain fluorophores challenging. For example, they use the Venus YFP fluorophore to visualize an activity dependant protein, Arc. The combination of a weak fluorophore and Arcs low-level baseline expression posed an imaging challenge.
The biggest differences we have seen [with the Prime BSI] are the increases in speed and signal to noise ratio.