Microprism-Mediated Calcium Imaging Reveals Neural Dynamics Over Time

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SEATTLE, April 10, 2024 — An approach to deep brain imaging developed at the University of Washington uses microprisms to provide stable tracking of neuronal activity over a large field of view. According to the research team, the microprism technique is a significant improvement over existing imaging methods used to characterize neuronal ensemble dynamics within deep brain tissues.

Organisms rely on their brains’ complex neural circuits to process information and perform functions that will help them find resources and avoid danger. Optical imaging techniques allow scientists to chart the changes to these circuits over time, to gain a clearer understanding of how new behaviors develop.

Fluorescence microscopy is used to track neuronal activity over time. Calcium indicators like GCaMP light up in response to calcium influx in the brain cells, and calcium levels rise when neurons fire. By averaging the fluorescence over time, scientists can use calcium indicators to map brain activity.
Using a microprism approach for brain imaging, researchers can track and map neural activity over days. Courtesy of Hjort et al., doi 10.1117/1.NPh.11.3.033407.
Using a microprism approach for brain imaging, researchers can track and map neural activity over days. Courtesy of Hjort et al., doi 10.1117/1.NPh.11.3.033407.

Calcium indicators can be excited by either single or multiphoton wavelengths of light. Multiphoton imaging results in sharper fields of view of the cell, which typically results in more accurate cell tracking. However, two-photon microscopy can only extend a few hundred μm from the brain surface. Without a reduction in resolution and yield, or the use of hyper-specialized dyes or equipment, two-photon microscopy and calcium indicators alone cannot visualize beyond the upper cortical layers of the brain.

Gradient-index (GRIN) lenses, which are implanted in the brain, enable researchers to track activity in deep regions of the brain. The imaging capabilities provided by GRIN lenses come with tradeoffs, though. Non-uniform excitation and fluorescence collection occur within the field of view, and warping can occur in visualized cellular morphology around the lens periphery.

In an alternative approach to deep brain imaging, the University of Washington team created a protocol for implanting microprisms to image deep brain regions and track neuronal activity, over multiple days, with high resolution and throughput.

The researchers performed two-photon calcium imaging through microprisms implanted in mouse brains and compared the yield, cell quality, and optical characteristics of their approach to the images obtained from standard GRIN lenses. Compared with GRIN lenses, the microprism-mediated calcium imaging method provided superior resolution quality and higher spatial resolution. The microprisms increased cell yield approximately 10x over GRIN lenses, in addition to providing a more uniform and much larger field of view.

The researchers visualized deep regions of the mouse brain via two microprisms 3 and 8 mm in length. Microprism-mediated calcium imaging of deep brain structures enabled the team to reliably track thousands of dynamic neurons across days within a single field of view in the deep brain regions.

The microprism-mediated calcium imaging approach facilitates longitudinal functional characterization of large groups of neural ensembles in vivo in cortical and subcortical brain structures. It achieves the necessary optical resolution for cellular, and potentially subcellular, imaging. Stable fields of view can be tracked over multiple sessions and stimulated using optogenetics.

Because microprisms use linear optics, excitation and morphology visualization are uniform across the field of view. Advances in microscope objectives have now made it possible to use microprisms for imaging subcortical regions, opening new possibilities for research.

“This approach represents a significant advancement in our ability to study how neural circuits evolve over time,” professor Garret D. Stuber said.

Using techniques like fluorescence microscopy and innovative tools like microprisms, scientists can gain new insights into how the brain adapts and changes over time. These advances are crucial for understanding fundamental processes underlying behavior and cognition.

The research was published in Neurophotonics (

Published: April 2024
A discipline that combines optics and genetics to enable the use of light to stimulate and control cells in living tissue, typically neurons, which have been genetically modified to respond to light. Only the cells that have been modified to include light-sensitive proteins will be under control of the light. The ability to selectively target cells gives researchers precise control. Using light to control the excitation, inhibition and signaling pathways of specific cells or groups of...
Fluorescence is a type of luminescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Specifically, fluorescence involves the absorption of light at one wavelength and the subsequent re-emission of light at a longer wavelength. The emitted light occurs almost instantaneously and ceases when the excitation light source is removed. Key characteristics of fluorescence include: Excitation and emission wavelengths: Fluorescent materials...
field of view
The field of view (FOV) refers to the extent of the observable world or the visible area that can be seen at any given moment through a device, such as an optical instrument, camera, or sensor. It is the angular or spatial extent of the observable environment as seen from a specific vantage point or through a particular instrument. Key points about the field of view include: Angular measurement: The field of view is often expressed in angular units, such as degrees, minutes, or radians. It...
Research & TechnologyeducationAmericasUniversity of WashingtonImagingLight SourcesOpticsoptogeneticslensesPrismsBiophotonicsmedicalGRIN lensesmicroprismsdeep brain imagingMicroscopymultiphoton microscopyfluorescenceneuroimagingcalcium imagingField of View

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