Our goal is to understand how stochasticity, non-linearity, correlations and coupled excited state dynamics of biological systems and networks contribute to their function. We investigate these questions in different systems and at different scales, from the fundamental principles of protein and bio-molecular dynamics on the nano-scale to the dynamic interactions in cellular networks giving rise to physiological function.

 To address these questions we take a multidisciplinary approach and develop new methods and technologies such as advanced imaging and spectroscopy techniques based on ultrafast and quantum optics and combine them with molecular biology, optogenetics and electrophysiology. We are currently pursuing three research areas:

Coupled Dynamics and Protein Function

Recent findings on the role of environmental vibrations in the observation of long-lived quantum coherences in energy transfer in the photosynthetic system point towards a more general realization that under certain biological conditions thermal environmental vibrations cannot be modeled as white noise. Vibrational correlations and coherences that arise due to structural constrains and system-bath coupling, could lead to stochastic resonances, non-Markovian dynamics or colored noise which may all contribute functionally beyond the context of quantum coherent energy transfer to the bio-molecular function at different scales.

We are investigating these general questions on the example of ion selectivity and transport in potassium channels. We are interested in understanding how the dynamic interactions in the selectivity filter are responsible for the observed high ion selectivity and transport rates and the possible functional role of coherent vibrational modes in this context.

Most of our today’s understanding on the function of channels is based on static crystallography data and electrophysiology. However, it is increasingly believed that in order to fully understand the mechanism that lead to the extreme ion selectivity and transport properties of ion channels, one has to account the fast dynamics of the system on the atomic scales.

To investigate these ideas, we are studying the dynamics of the selectivity filter using time resolved spectroscopy in the IR regime. Our goal is to experimentally identify the signatures of the transient interactions of K+ with binding sites of the selectivity filter during ion conduction and potential couplings that might lead to vibrational coherences.

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.