Light Beads Microscopy is a new method of two-photon microscopy optimized for volume imaging. It enables investigators to scan an entire volume in the rate that other conventional mesoscopes records just a single plane. This new technique makes use of columns of “Light Beads”, individual beams which are distinguishable in time and focus to different depths in the sample. Their novel approach uses 30 multiplexed beams, roughly an order of magnitude higher than any other previous temporal multiplexing approach demonstrated in vivo.  This quantum leap in imaging efficiency makes Light Beads Microscopy well suited for studying multi-regional encoding of sensory information and the dynamic interaction of brain networks at the single-neuron level.

Using Light Beads Microscopy and genetically encoded calcium indicators, Demas and colleagues imaged calcium transients in hundreds of thousands of cells in vivo, in portions of somatosensory-, visual-, posterior parietal-, and retro splenial–cortex, contained in a 3 mm X 5 mm area and a depth of 0.5 mm. The sampling rate of 5 Hz was per volume, not per plane. Stimuli used were whiskers perturbation and visual presentation of high-contrast drifting grates. Three sub-populations of neurons were identified that respond to whiskers stimuli, visual stimuli, or are spontaneously active. They also found evidence of mixed-selectivity in four anatomically separate clusters of cells, and of neurons that undergo distinct types of response modulation to one stimulus by the other located in separate anatomical locations.

To make the multiplexing happen, the laser light pulse is sent through two series of optical cavities that contain convex mirrors. The first cavity lets a small fraction of the energy of the laser pulse escape to the second cavity through a partially reflecting mirror (PRM) but sends the bulk of the laser energy back into the first cavity through a delay line loop created by the convex mirrors until it encounters the PRM again. The second cavity, which functions mainly as a delay line, splits the incoming pulse into two pulses. The first pulse is directed to the sample, while the second is delayed by the cavity before also being sent to the microscope. By travelling over and over in the first cavity and dividing pulses in the second one as described, a 90fs laser pulse is split into thirty ‘sub- pulses’ that occur only about 7ns apart from each other, with all 30 sub-pulses delivered to the sample in ~200ns.  MBF Bioscience engineers created the software for the Light Beads Microscope using ScanImage. This software was used to control the hardware and to receive and assemble the signals multiplexed in time and space from the photomultiplier tube.

Here at MBF Bioscience, thanks to a Small Business Innovation Research Grant from the NIMH, we are now working to commercialize this technology. We plan to optimize the hardware that creates the lights beads to reduce the overall microscope-system footprint and make it more versatile and easily adaptable to other laser-scanning microscopes and/or excitation wavelengths.

Light Beads Microscopy represents a major breakthrough in our ability to study the activity of large cell populations in the brain, and has the potential to revolutionize our understanding of how the brain encodes information.

Learn more about ScanImage and how it can help your research at: https://www.mbfbioscience.com/products/scanimage.

Reference:

Demas, J., Manley, J., Tejera, F. et al. High-speed, cortex-wide volumetric recording of neuroactivity at cellular resolution using light beads microscopy. Nat Methods 18, 1103–1111 (2021). https://doi.org/10.1038/s41592-021-01239-8

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Dr. Paul Manger’s new system developed by MBF Bioscience for analyzing large mammalian brain specimens

Dr. Paul Manger, Research Professor at the University of the Witwatersrand, South Africa, studies the brain anatomy of various animals to better understand the relationship between brain structure and function. Some of the mammals he studies have very large brains that don’t fit under a conventional research microscope. A team of scientists and microscopy experts at MBF Bioscience developed a new microscope system that enables Dr. Manger to analyze and image whole sections of large brain tissue. This system is the first for imaging and analyzing entire tissue sections of large mammalian brains. It will give Dr. Manger unprecedented analyses of African mammal brains and a way to share large images of tissue specimens with collaborators or a global audience.

To accommodate the large tissue specimens, MBF Bioscience developed the system with a Zeiss Axio Imager Vario microscope which is used in the semiconductor industry for inspecting silicon wafers. The motorized stage, built by Ludl, is enormous – it can hold tissue specimens with dimensions of up to 30cm x 40cm. This hardware, combined with Neurolucida, Stereo Investigator, and Biolucida software, allows Dr. Manger to capture 2D and 3D whole slide images of large tissue specimens, quantitatively analyze the specimens, and share the images with other scientists in a repository of African vertebrate brains. This type of imaging and analysis of large brains is not currently done in any other laboratory.

The motorized stage by Ludl can accommodate slides up to 30cm x 40cm

Dr. Manger now has the tools to help him uncover new information about the sensory, motor and cognitive capacities of African mammals. And, he has a new way to disseminate that information to scientists and collaborators who can build upon the findings to further the understanding of mammalian brains.

A sample of Dr. Manger’s publications can be found below.
Quantitative analysis of neocortical gyrencephaly in African elephants (Loxodonta africana) and six species of cetaceans: Comparison with other mammals
Organization and number of orexinergic neurons in the hypothalamus of two species of Cetartiodactyla: A comparison of giraffe (Giraffa camelopardalis) and harbour porpoise (Phocoena phocoena)
Neuronal morphology in the African elephant (Loxodonta africana) neocortex

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