Diffraction limited fluorescence microscopy has a resolution limit (Δx) that is imposed by the wavelength of excitation light (λ) and the numerical aperture of the objective (NA) such that
When imaging in live brain slices, it is usually necessary to use 2-photon microscopy with near-infrared excitation (λ=800 nm) as well as low numerical aperture water dipping objectives (NA=1). This imposes a diffraction limited resolution of ~400 nm, which is large compared to the size of many synaptic structures including the post-synaptic density (~300 nm) and the width of the neck of dendritic spines (~100 nm). Therefore the true structure of the synapse is obscured.
We have used stimulated-emission depletion microscopy (STED) to improve the resolution limit of 2-photon fluorescence microscopy to below the diffraction limit deep within brain tissue (see Ding et al). We are currently able to achieve ~100 nm resolution at depths of ~100 μm. Ongoing improvements in microscope design and the implementation of adaptive optics will likely improve the resolution to ~50 nm, allowing the accurate visualization of many synaptic and subcellular structures.
The use of photoactivatable (“caged”) glutamate has allowed the examination of signaling at individual visualized dendritic spines (see our papers by Carter, Bloodgood, Giessel, Busetto, Kwon…). Similarly, photoactivatable fluorophores provide a measure of protein trafficking in living cellular subcompartments (see Bloodgood, Steiner, Sturgill). We have developed microscopes and software to easily perform combined 2-photon laser photoactivation and 2-photon microscopy.
Furthermore, we are developing new photoactivatable molecules to examine modulation of neuron and synapse function by neuropeptides in the brain. These consist of short neuropeptides such as opioids and tachykinins which have been synthesized and attached to covalently linked function-blocking light-sensitive moieties. Brief flashes of ultaviolet (1-photon) or near infrared (2-photon) light can be used to photorelease active peptide with high spatial and temporal precision within brain tissue.
Fluorescence reporters of intracellular calcium have yielded tremendous insight into the mechanisms of calcium signaling and regulation in neurons. We are developing genetically-encoded fluorescence reporters to monitor the real-time activation of kinase and other signaling cascades within neurons. Fluorescence-lifetime imaging microscopy (FLIM) coupled with 2-photon fluorescence microscopy provides a quantitative measure of the state of neuronal and synaptic signaling cascades. We hope to eventually combine this approach with super-resolution microscopy to examine signaling microdomains.
July 15th, 2011