"This new imaging tool is expected not only to significantly promote our knowledge on brain function and its pathophysiology, but also accelerate development of novel therapies targeting neurological and neuropsychiatric disorders," said Daniel Razansky of the Helmholtz Center Munich and Technical University of Munich (TUM), who led the research. The research was carried out in a collaboration between the two institutes and Technion, the Israel Institute of Technology in Haifa.

To date, optical microscopy has been commonly used in studies of neuron activity, but it cannot penetrate deeper than a millimetre into the brain due to heavy scattering of the light. Larger-scale imaging techniques such as functional MRI are too slow to track neuron activity, which occurs in milliseconds. They also typically measure the haemodynamic response, an indirect effect of neuron activity on blood flow that only begins several seconds after activation.

Optoacoustic imaging can achieve high temporal resolution and penetrate centimetres of tissue. It already has several applications including measurements of tissue perfusion and oxygenation. By imaging animal models expressing the protein calcium sensor GCaMP5G, however, the technique can also detect neuronal activation. Calcium levels in neurons spike as they are activated, changing the sensor's optical absorbance within milliseconds and altering its acoustic output. The sensor was previously developed for fluorescence imaging.

Comparing the images

The FONT system comprises a pulsed laser that is tuneable to visible and near-infrared wavelengths and coupled with one of three hemispherical piezoelectric arrays designed to detect ultrasound signals in different brain volumes. The field-of-view and spatial resolution using the arrays can be varied between 50–1000 mm3 and 35–200 µm, respectively. The system has a temporal resolution of 10 ms. In their study, the researchers compared FONT images with fluorescence images also produced using the calcium indicator. To do so, they integrated the system with a sCMOS camera that was synchronized with the laser pulses.

In the first experiment, immobilised zebrafish larvae administered with a neuroactivating agent were imaged to provide the simplest scenario for proof of principle. Optoacoustic and fluorescent images correlated strongly (R2 = 0.97 and 0.98 in posterior and medial regions of the spinal cord). The researchers also successfully imaged freely moving larvae given the same neuroactivating agent, exploiting the high temporal resolution of the technique.

Fluorescence (top left) and FONT (top right) videos of an adult zebrafish following chemical neuroactivation. In the bottom panel, the grey 3D brain volume prior to activation has been reconstructed using optoacoustics. Activation signals reconstructed using FONT are superimposed onto the volume (scale bar 500 μm). CC BY 4.0 © Daniel Razansky et al 2016

In a third test, larger adult zebrafish brains measuring approximately 2 x 3 x 4 mm were excised and imaged. Using FONT, temporal variations in the protein signal in the volume were successfully reconstructed. Fluorescence images, however, were significantly degraded by the heavy scattering of light by the tissue.

Future goals

Advancing beyond the current study, the researchers' longer term goal is to image a whole brain in a mouse, as a more useful model for preclinical research. However, the larger volume of blood in rodents is more challenging. Haemoglobin is a strong absorber of the blue light used to detect GCaMP5G and produces a strong acoustic signal that swamps that produced by sensor.

Developing new sensors to operate at wavelengths not absorbed by blood is the key challenge that must be overcome before FONT can be fully exploited, co-author Gil Westmeyer, also of TUM and the Helmholtz Center Munich, told medicalphysicsweb. "In order to extend [FONT] from the zebrafish to the mouse, we need to push the molecular sensors into the near-infrared."

Westmeyer's group is one of several working on new sensors, developing both synthetic, chemical dyes that are injected locally into the brain and new genetic indicators that are encoded into animal models.

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