New technology has been developed at Tel Aviv University in Israel that significantly improves 2D and 3D imaging of neuronal activity in the brains of living animals. The technology should help make new findings possible about the workings of the brain and how neurological diseases operate.
PySight, as the technology is called, uses open-source software with commercially available hardware to give laser scanning microscopes a photon counting feature. Currently, a technique called multiphoton miscroscopy is used to see the short-lived events deep in the brain. In it, a laser excites fluorescent markers that were injected into the brain and these markers glow for a short time after excitation. Because there’s so much activity that happens at once, it’s hard to see much detail unless imaging is done with a very fast frame rate. But the faster the frame rate, the darker each frame becomes.
Photon counters are better in extremely dim situations, but commercially available devices aren’t meeting the needs of today’s neuroscientists. The Tel Aviv University team decided to built an affordable device that uses existing technology to provide a new capability for research.
Some details about the technology according to The Optical Society:
PySight provides high spatiotemporal resolution while producing a data stream that scales with the number of detected photons, not the volume or area being imaged. “Conventional data acquisition hardware stores the brightness of every pixel or 3D voxel even when it is zero because no photons were detected in that particular location,” [research team leader Pablo] Blinder explained. “PySight, however, only stores the precise detection time of each photon, allowing researchers to conduct rapid imaging of large volumes over long sessions, without compromising spatial or temporal resolution.”
To reconstruct a multidimensional image, knowing when each photon hits the detector isn’t enough. It’s necessary to also know where it originated in the brain. “This is even trickier if you want to simplify the system and avoid synchronizing the different scanning elements,” said Blinder. “To accomplish this, our software reads a list of photon arrival times along timing signals from the scanning elements, determines the origin of each photon within the sample and generates 3D movies that can span three or more dimensions.”
The photon arrival times are generated by a device known as a multiple-event time digitizer, or multiscaler, which records the times with a precision of 100 picoseconds. Another key component was an off-the-shelf resonant axial scanning lens that changes the focal plane hundreds of thousands of times per second. This lens was used to rapidly scan the laser beam across different depths within the brain and allowed the team to reconstruct continuous 3D images.
Here’s a couple of videos from the research demonstrating the new technique:
Using PySight, a combination of commercially available hardware and newly developed open-source software, the researchers performed rapid volumetric imaging of neurons and blood vessels in the brain of an intact, awake genetically-engineered larval zebrafish. The animal’s neurons expressed a genetically encoded fluorescent calcium indicator (GCaMP5g), and its red blood cells expressed a red fluorescent protein (DsRed). The total acquisition time of 134.2 seconds resulted in 7826 volume images. A resonant lens enabled axial scanning at 378 kHz. The imaged volume spanned 270 x 136 x 330 μm³.
Using PySight, a combination of commercially available hardware and newly developed open-source software, the researchers performed rapid volumetric imaging (about 30 volumes/second). of neurons in the brain of an awake genetically-engineered mouse. Its neurons expressed a genetically encoded fluorescent calcium indicator (GCaMP6f). To allow the display of volumes over time, data is aggregated in three channels correspond to depth between 0 to 90 μm, 90 to 180 μm and 180 to 270 μm for the red, green and blue channels correspondingly. The 60.24 seconds resulted in 1809 volume images acquired with the help of a resonant variofocal lens (TAG Lens 2.5) enabled axial scanning at 378 kHz. The imaged volume spanned 685 x 880 x 270 μm³.
Image: Brain vasculature in an anaesthetized mouse captured using Pysight. Credit: Pablo Blinder, Tel Aviv University
Study in Optica: PySight: plug and play photon counting for fast continuous volumetric intravital microscopy…
Via: The Optical Society…