Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency

Maioli, Vincent; Boniface, Antoine; Mahou, Pierre; Ortas, Júlia Ferrer; Abdeladim, Lamiae; Beaurepaire, Emmanuel; Supatto, Willy

doi:10.1364/boe.400113

Cited by 32 publications

(18 citation statements)

References 41 publications

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“…3). Similar improvement is obtained with second harmonic generation 5 . Due to its higher order, even stronger enhancement (~64) is reached with three-photon excited fluorescence (Fig.…”

Section: Dyesupporting

confidence: 83%

“…However, this signal enhancement is limited by potential nonlinear photodamage due to high peak intensities. To characterize the threshold of photodamage, we then used heartbeat rate as a sensitive reporter of linear and nonlinear disruptions 5 . By analyzing 500 experimental conditions in 100 embryos, we established the scaling law governing highly nonlinear photodamage (Fig.…”

Section: Dyementioning

confidence: 99%

“…1c). In addition, we quantified linear effects such as heating due to water absorption and photobleaching 5 . Together, these analyses established the signal enhancement limit, which is reachable by adjusting the laser repetition rate f=1/T at a given mean power (Fig.…”

Section: Dyementioning

confidence: 99%

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Fast in vivo multiphoton microscopy using optimized light-sheet illumination

Maioli

Boniface

Mahou

et al. 2021

Multiphoton Microscopy in the Biomedical Sciences XXI

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Light-sheet fluorescence microscopy is a method of choice for multiscale live imaging. Indeed, its orthogonal geometry results in high acquisition speed, large field-of-view and low photodamage. Its combination with multiphoton excited fluorescence improves its imaging depth in biological tissues. However, it appears femtosecond laser sources commonly used in multiphoton microscopy at an 80 MHz repetition rate may not be optimized to take full advantage of light-sheet illumination during live imaging. Hence, we investigated the nature of induced photodamage in multiphoton light-sheet microscopy and the influence of laser parameters on the signal-to-photodamage ratio. To this end, we used zebrafish embryonic heart beat rate and fluorophore photobleaching as sensitive reporters of photoperturbations. We characterized linear and nonlinear disruptions depending on laser parameters such as laser mean power, pulse frequency or wavelength, and determine their order and relative impact. We found an optimal pulse frequency of ~10 MHz for imaging mCherry labeled beating hearts at 1030 nm excitation wavelength. Thus, we achieved high-speed imaging without inducing additional linear heating or reaching nonlinear photodamage compared to previous implementation. We reach an order-ofmagnitude enhancement in two-photon excited fluorescence signal by optimizing the laser pulse frequency while maintaining low both the laser average power and its peak irradiance. It is possible to reach even larger enhancement of 3photon excited fluorescence using such laser parameters. More generally, using low laser pulse frequency in multiphoton light-sheet microscopy results in a drastic improvement in signal level without compromising live sample, which opens new opportunities for fast in vivo imaging.

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“…3). Similar improvement is obtained with second harmonic generation 5 . Due to its higher order, even stronger enhancement (~64) is reached with three-photon excited fluorescence (Fig.…”

Section: Dyesupporting

confidence: 83%

Section: Dyementioning

confidence: 99%

See 1 more Smart Citation

Fast in vivo multiphoton microscopy using optimized light-sheet illumination

Maioli

Boniface

Mahou

et al. 2021

Multiphoton Microscopy in the Biomedical Sciences XXI

Self Cite

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“…The use of two-photon (2P) excitation 16 instead, employing near infrared (IR) pulsed light, which is invisible to the majority of vertebrates (zebrafish included 17,18 ), allows for a potential expansion of the experimental panorama amenable to high-speed imaging. However, owing to the low probability of the 2P absorption process, 2P LSFM is prone to low signal-to-noise ratio (SNR) issues, which remarkably limit the actual volumetric acquisition speed 19 . For this reason, so far, 2P LSFM has been proficiently applied to perform structural studies 20 , single plain functional imaging 21,22 and proof-of-principle functional studies at low volumetric rate 23 .…”

Section: Introductionmentioning

confidence: 99%

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

Vito

Turrini

Müllenbroich

et al. 2022

Biomed. Opt. Express

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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. At a moderate PTZ concentration we observed a widespread increase in nervous activity synchronization with the progression of the exposure time to the drug, particularly in the optic tectum, associated with an unexpected synchronization decrease happening in small spatially-defined areas. At saturating PTZ concentrations we instead observed a brain-wide functional connectivity reorganization and the emergence of distinctive phases of ictal and postictal activity. During the ictal phase the degree of synchronization in the neuronal activity dramatically increases in the whole-brain. On the contrary, the degree of neuronal synchrony in the postictal phase resembles the control condition, with the exception of the dorsal thalamus, where it increases, and subregions of the spinal cord, where it unexpectedly decreases.The volumetric frame rate of our system allowed us to observe the emergence of previously unreported fast rhythmic ictal waves propagating in postero-anterior direction, which we termed caudo-rostral ictal waves (CRIWs), spanning across the whole larval encephalon in about 1 s during the ictal phase.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.

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“…However, it still has limited FOV (340 × 650 µm) and depth (135 µm), which restricted the analysis to only the motor cortex [ 137 ]. In vivo two-photon light-sheet imaging [ 135 , 136 , 138 , 139 ] has not yet been applied for wide-field imaging in mice or rats. However, taking into account the rapid technological advances in multiphoton imaging, we anticipate that the combination of a two-photon setup and light-sheet microscopy will soon be implemented to further increase the FOV of functional fluorescent imaging.…”

Section: Introductionmentioning

confidence: 99%

Emerging imaging methods to study whole-brain function in rodent models

et al. 2021

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In the past decade, the idea that single populations of neurons support cognition and behavior has gradually given way to the realization that connectivity matters and that complex behavior results from interactions between remote yet anatomically connected areas that form specialized networks. In parallel, innovation in brain imaging techniques has led to the availability of a broad set of imaging tools to characterize the functional organization of complex networks. However, each of these tools poses significant technical challenges and faces limitations, which require careful consideration of their underlying anatomical, physiological, and physical specificity. In this review, we focus on emerging methods for measuring spontaneous or evoked activity in the brain. We discuss methods that can measure large-scale brain activity (directly or indirectly) with a relatively high temporal resolution, from milliseconds to seconds. We further focus on methods designed for studying the mammalian brain in preclinical models, specifically in mice and rats. This field has seen a great deal of innovation in recent years, facilitated by concomitant innovation in gene-editing techniques and the possibility of more invasive recordings. This review aims to give an overview of currently available preclinical imaging methods and an outlook on future developments. This information is suitable for educational purposes and for assisting scientists in choosing the appropriate method for their own research question.

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Fast in vivo multiphoton light-sheet microscopy with optimal pulse frequency

Cited by 32 publications

References 41 publications

Fast in vivo multiphoton microscopy using optimized light-sheet illumination

Fast in vivo multiphoton microscopy using optimized light-sheet illumination

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy

Emerging imaging methods to study whole-brain function in rodent models

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