Deciphering the workings of a brain (and its alterations due to pathologies and disorders) requires real-time mapping of the activity of all neurons. Recently, this formidable task has been brought into reach by the implementation of light sheet fluorescence (LSF) microscopy on the transparent zebrafish larva, a simple yet representative model of the vertebrate brain. This application is however hindered by the use of visible light for fluorescence excitation , inducing unwanted visual stimulation that can only be partially overcome resorting to complex excitation geometries. On the other hand, two-photon (2P) LSF microscopy, owing to the use of invisible infra-red illumination, enables volumetric imaging of neuronal activity avoiding undesirable visual stimulation . However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited (~ 1 Hz) by the signal to noise ratio achievable without sample photodamage. Here we describe a custom-made 2P LSF microscope that overcomes this limit, being specifically designed to perform whole-brain cellular-resolution calcium imaging in zebrafish larvae quintuplicating previous volumetric acquisition frequency (5 Hz) . ...%DIFDELCMD < ] %%% We employed this system to investigate, in real-time on a brain-wide scale, the onset and propagation of acute seizures, as induced by the convulsant drug pentylenetetrazol (PTZ), avoiding detrimental visual stimulation on a highly susceptible system such as an epileptic brain. ...%DIFDELCMD < ] %%% In conclusion, the presented 2P LSF microscope design affords high spatio-temporal resolution while avoiding visual stimuli and allows unprecedented access to whole zebrafish brain epileptic dynamics .
Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics . However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) %DIFDELCMD < ] %%% while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, %DIFDELCMD < ] %%% characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs) .
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Deciphering the workings of a brain (and its alterations due to pathologies and disorders) requires
Light-sheet fluorescence microscopy (LSFM) enables
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mapping of the activity of all neurons. Recently, this formidable task has been brought into reach by the implementation of light sheet fluorescence (LSF) microscopy on the transparent zebrafish larva, a simple yet representative model of the vertebrate brain. This application is however hindered by
whole-brain functional imaging in zebrafish larvae. Conventional one photon LSFM can however induce undesirable visual stimulation due to
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light for fluorescence excitation , inducing unwanted visual stimulation that can only be partially overcome resorting to complex excitation geometries. On the other hand,
excitation light. The use of
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LSF microscopy, owing to the use of invisible infra-red illumination, enables volumetric imaging of neuronal activity avoiding undesirable visual stimulation
excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics
R
(~ 1 Hz) by the signal to noise ratio achievable without sample photodamage. Here
by the signal-to-noise-ratio. Here,
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custom-made
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LSF microscope that overcomes this limit, being specifically designed to perform whole-brain cellular-resolution calcium imaging in zebrafish larvae quintuplicating previous volumetric acquisition frequency
LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain
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.
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...
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We employed this system to investigate, in real-time on a brain-wide scale, the onset and propagation of
while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced
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as induced by the convulsant drug pentylenetetrazol (PTZ), avoiding detrimental visual stimulation on a highly susceptible system such as an epileptic brain.
D
...
R
In conclusion, the presented 2P LSF microscope design affords high spatio-temporal resolution while avoiding visual stimuli and allows unprecedented access to whole zebrafish brain epileptic dynamics
characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs)