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In vivo quantification of neuronal activity in zebrafish with ORCA®-Quest
Capturing high quality images of live organisms, where changes happen almost instantly is an intricate observation, which presents many challenges.
Presently doing research at the Max Planck Institute for Biological Cybernetics in Germany, Dr. Drew Robson methodically develops the different approaches to observe the zebrafish larvae brain function during its natural behavior with a quantitative photon number-resolving camera.
From zebrafish brains to human brains
Zebrafish in aquarium
Zebrafish are an emerging model organism to study brain activities. In fact, zebrafish and humans share many commonalities making them ideal to help us understand our brain function, health and disease. Drew Robson, PhD. from Harvard University, currently studies these fish in their larvae stage. During this time, the fish is transparent making it easier to observe directly at any cell in the body while it is growing.
To enable the endeavor of observing the larvae in its swimming state, the researcher devised a closed-loop system, which tracks and corrects its position by keeping the brain in the microscope field of view. The experimental undertaking of investigating its natural behavior is complex and requires the right methods.
Microscopy challenges
To grasp the ephemeral changes occurring in the larvae brain requires detailed high-resolution imaging in space and time at a subcellular resolution. Imaging of live organism is not easily accessible and poses many challenges. In order to minimize aberrations and noise influences, this experiment required using a light sheet fluorescence microscope, and a customized setup with well-designed optics and detection capabilities.
Rahul Trivedi, Postdoctoral Researcher at Max Planck Institute at the microscope
The source of signal within the zebrafish brain is a fluorescent protein calcium sensor. Observing fluorescence signals consists of a series of compromises. For the signal itself, a high-excitation energy provides the best signal to noise ratio. However, an increased excitation energy also damages and disturbs the normal brain function, potentially creating a bleach risk of the fluorescent protein and limiting prolonged observation.
Additionally, as neuronal signaling happens on short timescales, the exposure time must be low, enhancing the aforementioned conflict. In the end, the output signal-to-noise ratio and the excitation energy must be carefully balanced for each experiment to provide the best possible result.
Quantification of neuronal activity
Volume render of pan-neuronal H2B-GCaMP6s (green) and ReaChR in Islet2b (taken with the ORCA-Quest)
To address this sensitive balance, Dr. Robson acquired a quantitative photon number-resolving camera, first in the world to meet such demands. This detection camera known under the name ORCA-Quest qCMOSⓇ camera (for quantitative CMOS) eased a great deal of the conflict presented between excitation and SNR.
The low-readout noise, combined with the high speed of the ORCA-Quest enabled observation of the zebrafish brain in three dimensions over a prolonged period of time.
Even in the very low signal regimes, where the naturally occurring shot noise of photons becomes apparent, the ORCA-Quest provides linear results enabling quantification of neuronal activity. The format of the sensor combined with the high-pixel number allows dual channel observation across a whole zebrafish brain at subcellular resolution.
Whereas large format and high-resolution sensors have been available on the scientific camera market, no option so far was reaching Dr. Robson’s high requirements concerning noise levels, a crucial component in their detection and computational reconstruction workflow. In addition, the ORCA-Quest offered them unprecedented frame rates without compromising the noise floor or resolution.
Maximum intensity projection of pan-neuronal H2B-GCaMP6s (green) and ReaChR in Islet2b neurons (red) (taken with the ORCA-Quest)
About Dr. Drew Robson
Dr. Drew Robson received his B.A. in Mathematics from Princeton University, where he also worked on computational biology and biophysics in the labs of Olga Troyanskaya and Eric Wieschaus. He received his Ph.D. from Harvard University, where he worked on thermosensory behaviors and brain-wide neural imaging in the Schier and Engert labs. He led a joint lab in Systems Neuroscience and Neuroengineering with Jennifer Li at the Rowland Institute at Harvard from 2014 to 2019. He moved his lab from the Rowland Institute to the Max Planck Institute for Biological Cybernetics in 2019.
Product used in this case study
The C15550-20UP is the world's first camera to incorporate the qCMOS image sensor. The camera achieves the ultimate in quantitative imaging.
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