We aim to understand biological function on a multi-level scale: the fundamental principles of protein and bio-molecular dynamics on the nano-scale, and the dynamic interactions in cellular networks giving rise to macroscopic function and behavior on the organism level. The success of this endeavor will critically depend on the development of new techniques and approaches, which represents a main focus of our research.

Biological Function at the Quantum Limit

Has nature evolved in a way to be able to take advantage of any quantum mechanical effects in biological function? We are interested to answer this question experimentally by pushing the real time mechanistic understanding of function of ion channels and pumps as nano-scale “protein machines” working at their structural and energetic limits.

We aim to answer open, fundamental questions regarding the mechanisms that lead to the high level of specificity, directedness and efficiency of the ion selectivity and transport and discover to what extent quantum coherence and its interplay with environmental noise is responsible for these observed functional properties (Figure 1).

It is now believed that to understand ion selectivity and transport, one has to account for competing microscopic interactions for whose quantitative description, accurate models on the atomic level are required. In this context our quantum mechanical model and recent theoretical results have shown that on this scale quantum coherence should be involved in the selectivity and transport process. We expect these coherences to shine new light on some of the above questions (Figure 2).

High resolution optogenetic mapping of neuronal circuits

The ability to stimulate neuronal activity in a non-invasive and cell specific fashion is necessary to study a wide range of fundamental neuroscience questions. The recently discovered class of genetically expressible photoactivatable ion-channels such as Channelrhodopsin has enabled the optical control of neural activity. The most widely used approach has been the optical activation of the genetically expressed light-gated ion-channel, Channelrhodopsin-2 (ChR2), to initiate population activity in neuronal circuits. However, given the low channel conductance, the initiation of action potentials is only possible when a sufficiently large number of channels are activated at the same time, which has made single cell resolution of optogenetic activation a major challenge.

To overcome these limitations, we have recently developed a scheme for fast, selective and targeted control of neuronal activity with single cell resolution in mouse and rat hippocampal slices. Using the scanningless technique of temporal focusing for which the axial beam profile can be controlled independently of its lateral distribution, large number of channels on individual neurons can be excited simultaneously leading to strong (up to 15mV) and fast (≤1ms) depolarisations. It is further shown that cellular compartments such as dendrites and large presynaptic terminals can be activated selectively at depths up to 150mm. (Figure 3)

We have further shown that sub-cellular compartments such as dendrites and large presynaptic terminals can be activated by this technique. The spatial and temporal resolution provided by this technique allows for fine manipulation of neuronal activity to study and control the function of neuronal microcircuits in vitro and in vivo. In particular in the context of neuronal circuit mapping and dendritic integration it can be used as a tool for high throughput mapping of connectivity and provides the means to study mechanism of neuronal input integration on a single neuron with unprecedented spatiotemporal resolution.

Multilayer three-dimensional super-resolution microscopy of cellular protein distributions

Photoactivated Localization Microscopy (PALM) is one of the recently emerging techniques for optical imaging of protein distributions in biological samples at nanometer resolution. In PALM, numerous sparse subsets of photoactivatable fluorescent proteins are serially activated, localized, and bleached. The aggregate position information from all subsets is then assembled into a super-resolution image. In its initial realization, PALM used total internal reflection excitation (TIRF), thus the technique was limited to thin biological samples close to a microscope coverslip surface.

 
By combing PALM with temporal focusing, we have demonstrated that protein distributions deep (~10 μm) in cells with a lateral localization precision better than 50nm could be imaged. Temporal focusing is a nonlinear optical technique whereby ultra-short laser pulses are focused in time. Using diffraction gratings and an appropriate geometry, it is possible to focus an optical pulse in the axial direction without focusing it in the lateral directions. Therefore, high spatial energy densities can be created in a widefield configuration, which can be used for two-photon excitation of fluorescent proteins. Using genetically expressed fluorescent proteins, this technique permits the study of protein-protein and other biological interactions at high resolutions in vivo. We have imaged amongst others proteins in the mitochondrial matrix and have resolved the cell membrane structure of Drosophila S2 cells in 3D. (Figure 4).

For more details of the Vaziri group's research, please visit their website.