Electroporation of Ca dyes in vivo.

A collection of scientists at Yale and Huazhong Universities have developed a method for loading of neurons in vivo. No great shakes you might say? Well it turns out they have managed to load dextran-conjugated fluorescent Ca dyes into neurons so that one can see Ca dynamics in spines heads! The figure is 6C from their paper showing spines and line-scan imaging of electrically evoked fluorescence changes. I have to point out they need to average 8 traces to this poor S/N, even with a high affinity Ca indicator (Kd 200 nM, 0.02 mM). I would think this dye would be saturated within the spine head, but clearly it is not (labs that patch clamp cells ex vivo use lower affinity dyes such as fluo-4FF (0.15-0.5 mM), or OGB-5N (0.5 mM) but see much stronger signals during synaptic inputs).

These quibles aside, This is important technical accomplishment, as it is the first report of imaging spines heads so clearly with a Ca dye. They also claim to image boutons, which is even more amazing. Frankly, I am not used to looking at boutons in vivo (or ex vivo) so I find those data hard to judge. The spines look lovely though.

In Vivo Simultaneous Tracing and Ca Imaging of Local Neuronal Circuits.
Nagayama, et al., Neuron (2007) 53:789-803. DOI 10.1016/j.neuron.2007.02.018

The Munich group published a very nice paper on their technique last year:

Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo.
O Garaschuk, R-I Milos, A Konnerth Nature Protocols (2006)
1:380-386.