We have synthesized a 7-diethylaminocoumarin (DEAC) derivative that allows wavelength selective, two-photon uncaging at 900 nm versus 720 nm. This new caging chromophore, called DEAC450, has an extended π-electron moeity at the 3-position that shifts the absorption spectrum maximum of DEAC from 375 nm to 450 nm. Two-photon excitation at 900 nm was more than 60-fold greater than at 720 nm. Two-photon uncaging of DEAC450-Glu at 900 nm at spine heads on pyramidal neurons in acutely isolated brain slices generated postsynaptic responses that were similar to spontaneous postsynaptic excitatory miniature currents, whereas significantly higher energies at 720 nm evoked no currents. Since many nitroaromatic caged compounds are two-photon active at 720 nm, optically selective uncaging of DEAC450-caged biomolecules at 900 nm may allow facile two-color optical interrogation of bimodal signaling pathways in living tissue with high resolution for the first time.

The autism-spectrum disorder Tuberous Sclerosis Complex (TSC) is caused by mutations in the Tsc1 or Tsc2 genes whose protein products form a heterodimeric complex that negatively regulates mTOR-dependent protein translation. Although several forms of synaptic plasticity, including metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD), depend on protein translation at the time of induction, it is unknown if these forms of plasticity require signaling through the Tsc1/2 complex. To examine this possibility, we postnatally deleted Tsc1 in vivo in a subset of hippocampal CA1 neurons using viral delivery of Cre recombinase in mice. We found that hippocampal mGluR-LTD was abolished by loss of Tsc1, whereas a protein synthesis-independent form of NMDA receptor-dependent LTD was preserved. Additionally, AMPA and NMDA receptor mediated excitatory postsynaptic currents (EPSCs) and miniature spontaneous EPSC frequency were enhanced in Tsc1 KO neurons. These changes in synaptic function occurred in the absence of alterations in spine density, morphology, or pre-synaptic release probability. Our findings indicate that signaling through Tsc1/2 is required for the expression of specific forms of hippocampal synaptic plasticity as well as the maintenance of normal excitatory synaptic strength. Furthermore, these data suggest that perturbations of synaptic signaling may contribute to the pathogenesis of TSC.

SUMMARY Two-photon laser scanning microscopy (2PLSM) has allowed unprecedented fluorescent imaging of neuronal structure and function within neural tissue. However, the resolution of this approach is poor compared to that of conventional confocal microscopy. Here we demonstrate supraresolution 2PLSM within brain slices. Imaging beyond the diffraction limit is accomplished by using near-infrared (NIR) lasers for both pulsed 2-photon excitation and continuous wave stimulation emission depletion (STED). Furthermore, we demonstrate that Alexa Fluor-594, a bright fluorophore commonly used for both live cell and fixed tissue fluorescence imaging, is suitable for STED 2PLSM. STED 2PLSM supraresolution microscopy achieves approximately 3 fold improvement in resolution in the radial direction over conventional 2PLSM, revealing greater detail in the structure of dendritic spines located ~100 microns below the surface of brain slices. Further improvements in resolution are theoretically achievable, suggesting that STED 2PLSM will permit nanoscale imaging of neuronal structures located in relatively intact brain tissue.

Two-photon laser scanning microscopy (2PLSM) allows fluorescence imaging in thick biological samples where absorption and scattering typically degrade resolution and signal collection of one-photon imaging approaches. The spatial resolution of conventional 2PLSM is limited by diffraction, and the near-infrared wavelengths used for excitation in 2PLSM preclude the accurate imaging of many small subcellular compartments of neurons. Stimulated emission depletion (STED) microscopy is a superresolution imaging modality that overcomes the resolution limit imposed by diffraction and allows fluorescence imaging of nanoscale features. Here, we describe the design and operation of a superresolution two-photon microscope using pulsed excitation and STED lasers. We examine the depth dependence of STED imaging in acute tissue slices and find enhancement of 2P resolution ranging from approximately fivefold at 20 μm to approximately twofold at 90-μm deep. The depth dependence of resolution is found to be consistent with the depth dependence of depletion efficiency, suggesting resolution is limited by STED laser propagation through turbid tissue. Finally, we achieve live imaging of dendritic spines with 60-nm resolution and demonstrate that our technique allows accurate quantification of neuronal morphology up to 30-μm deep in living brain tissue.

The structure of dendritic spines suggests a specialized function in compartmentalizing synaptic signals near active synapses. Indeed, theoretical and experimental analyses indicate that the diffusive resistance of the spine neck is sufficient to effectively compartmentalize some signaling molecules in a spine for the duration of their activated lifetime. Here we describe the application of 2-photon microscopy combined with stimulated emission depletion (STED-2P) to the biophysical study of the relationship between synaptic signals and spine morphology, demonstrating the utility of combining STED-2P with modern optical and electrophysiological techniques. Morphological determinants of fluorescence recovery time were identified and evaluated within the context of a simple compartmental model describing diffusive transfer between spine and dendrite. Correlations between the neck geometry and the amplitude of synaptic potentials and calcium transients evoked by 2-photon glutamate uncaging were also investigated.

Two-photon fluorescence microscopy has been used extensively to probe the structure and functions of cells in living biological tissue. Two-photon excitation generates fluorescence from the focal plane, but also from outside the focal plane, with out-of-focus fluorescence increasing as the focus is pushed deeper into tissue. It has been postulated that the two-photon depth limit, beyond which results become inaccurate, is where in-focus and out-of-focus fluorescence are equal, which we term the balance depth. Calculations suggest that the balance depth should be at ϳ600 m in mouse cortex. Neither the two-photon depth limit nor the balance depth have been measured in brain tissue. We found the depth limit and balance depth of two-photon excitation in mice with GCaMP6 indicator expression in all layers of visual cortex, by comparing near-simultaneous two-photon and three-photon excitation. Two-photon and three-photon results from superficial locations were almost identical. two-photon results were inaccurate beyond the balance depth, consistent with the depth limit matching the balance depth for two-photon excitation. However, the two-photon depth limit and balance depth were at 450 m, shallower than predicted by calculations. Our results were from tissue with a largely homogenous distribution of fluorophores. The expected balance depth is deeper in tissue with fewer fluorophores outside the focal plane and our results therefore establish a superficial bound on the two-photon depth limit in mouse visual cortex.

Random scattering of light by a turbid layer prevents conventional imaging of objects hidden behind it. Angular correlations in the scattered light, created by the so-called optical memory effect, have been shown to enable computational image retrieval of hidden sources. However, basic memory-effect imaging contains no spatial (x) information, as only angular (k-space) measurements are made. Here, we use windowed Fourier transforms to record scattered-light images in the full {x,k} phase space. The result is the ability to discriminate size and depth of individual sources that are hidden behind a thin scattering layer.

Highlights d DS/OS and LS axons form two major functional types of inputs from dLGN to V1 d Both DS/OS and LS boutons are distributed from superficial to middle layers in V1 d Single-axon arbors are reconstructed for DS/OS and LS axons d DS/OS axons have denser arbors in the middle layers than LS axons