Measuring the faintest and most phototoxicity sensitive processes in the brain.
The axonal arborization made from fine and thin processes is extremely hard to fill with fluorescent dyes. On the other hand, these structures are extremely sensitive, and phototoxicity arrives in an unexpected way: it first appears to affect ultrastructure, and only at larger extents alter functional mesaurements: Ca2+ transients. See supplementary data of Noemi Holderith, Andrea Lorincz, Gergely Katona, Balázs Rózsa, Akos Kulik, Masahiko Watanabe & Zoltan Nusser Release probability of hippocampal glutamatergic terminals scales with the size of the active zone, Nature Neuroscience (2012)
As a result, to image structures faint and sensitive like axons and boutons, topmost quality detection optics and detectors are necessary. Our travelling detector assemblies equipped with GaAsP detector modules reach to the physical limits of detection efficiency.
Top, CA1 hippocampal interneuron filled with 100µM Alexa-594 and 60 µM Fluo-4 in a rat acute brain slice. Boxes show successive zooming onto one axonal segment, showing presynaptic boutons. Bottom left, red and green channel raw traces of the line-scan along the axon.
Bottom right, Ca2+ responses from four of the synaptic input locations. Ca2+ transient calculated from the marked region is plotted with blue.
Images courtesy of Aniko Vágvölgyi, Institute of Experimental Medicine, Hungarian Academy of Sciences