ScienceWise - Jul/Aug 2009

The Holographic Neurone Stimulator

When physics and biology combine

For some time neuroscientists have been using microscopic electrodes to excite nerve cells in order to study their response to various stimulation patterns and to unravel the secrets of how nerves process information. However, inserting an electrode into a dendrite only a few µm thick is a very difficult task. Doubly so if you require multiple points to be stimulated at the same time. This approach is also slow and painstaking so you can’t really select and excite a sequence of contact points anywhere near as fast as it happens in living neural networks.

This reliance on electrodes has posed some limitations on the types of experiments neurobiologists have been able to conduct. However, two neurobiologists, Dr. Christian Stricker and Prof. Steve Redman of the John Curtin School of Medical Research, have recently achieved a breakthrough in this area in a collaborative project with physicists at the ANU Department of Quantum Science.

“We were looking for a system that could generate real time images of living neurones in three spatial dimensions and then stimulate those neurones at several specific points.” Dr Stricker explains. “So we approached physicists Professor Hans Bachor and Dr. Vincent Daria to explore what we might be able to achieve collaboratively.” As often happens with collaborations, experts from diverse fields were able to pool their expertise and create a system that none of them could have built individually. The result was a new tool in neuroscience which the team have christened the Holographic Neuron Stimulator.

The Holographic Neuron Stimulator works by immersing a sample of living neurones in a solution containing neurotransmitters - a class of  molecules that stimulate neuronal  firing. Of course if the cells were simply bathed in active neurotransmitters they would fire constantly. So scientists have adapted a “caged” neurotransmitter molecule such that it only becomes active (or “uncaged”) in the presence of a strong light field.

In order for the system to work effectively, the triggering light has to be highly localised at selected points in space. The team decided that the best way to achieve this was to use a holographic projection technique.

A normal photographic hologram is a combination of dense and transparent regions in a photographic emulsion that don’t outwardly look like anything recognisable. But when illuminated by a broad plane coherent wave, such as that produced by an expanded laser beam, the dense and transparent regions in the hologram project an interference pattern that mimics an object in 3 dimensional space. In conventional holography, the hologram is recorded on a photographic plate using the reverse process – laser illumination of a real object and interference with a second beam.  Although many holograms are recorded in this photographic way, it’s quite possible to calculate the holographic pattern of an object using optical theory alone. Such pre-calculated holographic patterns are commonly called Computer Generated Holograms (CGH).  A programmable electronic light modulator can be encoded with such a CGH, and project a complex three dimensional  light pattern from a single laser.

The projected light pattern from the hologram can be in the form of tiny spots of light, which could in principle be used to create bright spots within sections of neural tissue. If that tissue were surrounded by an inactive “caged” neurotransmitter solution, the holographically projected bright spots would release (or uncage) the transmitters at various points in the sample. If those points were made to correspond with the location of a nerve cell membrane, the result would be to stimulate the cell and potentially initiate a nerve impulse.

This is precisely what the Holographic Neuron Stimulator does. Using a programmable hologram to alter the shape of the laser beam and a powerful computer, the machine creates a series of patterns of spots in precisely determined locations for stimulating various sections in a neuron. This is more versatile than using a simple mask or lens.  Another advantage is that it can be changed in real time allowing the light spots to be switched and moved every few milliseconds. In this way scientists can stimulate several points on the same neurone either simultaneously or in a set temporal sequence.
A significant challenge with any optical neurone stimulating system is correlating your light spots with features on the actual neurones in the sample. The Holographic Neuron Stimulator achieves this by using the same holographic technique to create a special kind of microscope known as multi-photon fluorescence microscope or MFM.

An MFM works by using a femtosecond-pulse laser to excite natural molecules in the sample into fluorescence. The simplest kind of fluorescence is when a molecule absorbs a highly energetic photon and re-emits a less energetic one. This is commonly seen when things glow under ultraviolet light. This isn’t very useful in microscopy as it would cause the entire sample to absorb light and glow at once. So the fluorescence event employed by a MFM is the absorption of two or more low-energy infrared photons to excite one molecule, which then emits in the visible spectrum. Because of quantum rules, in order to raise the energy in two jumps, both photons must be absorbed by the molecule at exactly the same time. Hence, to increase the probability of simultaneous multi-photon absorption, the density of photons at an instant of time needs to be very high, which can only be achieved in a strongly focussed pulsed-laser with pulse-width in the order of several femtoseconds (10-15 s.)

Prior to using the Holographic Neuron stimulator to excite impulses, a 3D image of the neuron sample is created by switching the system to MFM mode. By raster scanning the femtosecond-pulse laser beam across the sample very quickly, a beautiful crisp three dimensional image of the neuron is generated.  Once the 3D image of the neuron is acquired, the hologram for projecting the appropriate light spot pattern is calculated and encoded on the programmable hologram. 
To a neuroscientist trying to understand how billions of individual neurones integrate together to create complex structures like the human brain, this new technique offers a very exciting opportunity to do new science.  “The great thing about this set up is that you can generate an image of a living neurone in situ, identify points that you wish to stimulate, then switch to stimulate mode and directly hit those points in any sequence you like. “ Dr Stricker says. “In neuroscience we are always looking to push the boundaries and this should really help us do so.” He is looking forward to the first trial runs of the stimulator.

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