Simultaneous Two- and Three-photon Imaging of Multilayer Neural Activities with Remote Focusing

Mok, Aaron T.; Wang, Tianyu; Xia, Fei; Wu, Chuanbin; Xu, Chris

doi:10.1364/cleo_at.2019.am1i.5

Cited by 7 publications

(6 citation statements)

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“…Together our results demonstrate that although both 2P and 3P excitation can be used for cuticle through imaging at the superficial layers of the fly brain such as the mushroom body, 3P outperforms 2P in deeper brain regions such as the central complex especially when cellular and subcellular resolution is necessary. This conclusion is consistent with the imaging studies conducted in the mouse brain (Mok et al, 2019;Wang et al, 2018a;Wang et al, 2020).…”

Section: P/3p Imaging Of Mushroom Body and Central Complex Neurons Th...supporting

confidence: 92%

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle

Aragon

Mok

Shea

et al. 2022

eLife

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We developed a multiphoton imaging method to capture neural structure and activity in behaving flies through the intact cuticles. Our measurements show that the fly head cuticle has surprisingly high transmission at wavelengths > 900 nm, and the difficulty of through-cuticle imaging is due to the air sacs and/or fat tissue underneath the head cuticle. By compressing or removing the air sacs, we performed multiphoton imaging of the fly brain through the intact cuticle. Our anatomical and functional imaging results show that 2- and 3-photon imaging are comparable in superficial regions such as the mushroom body, but 3-photon imaging is superior in deeper regions such as the central complex and beyond. We further demonstrated 2-photon through-cuticle functional imaging of odor-evoked calcium responses from the mushroom body g-lobes in behaving flies short-term and long-term. The through-cuticle imaging method developed here extends the time limits of in vivo imaging in flies and opens new ways to capture neural structure and activity from the fly brain.

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Section: P/3p Imaging Of Mushroom Body and Central Complex Neurons Th...supporting

confidence: 92%

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle

Aragon

Mok

Shea

et al. 2022

eLife

Self Cite

View full text Add to dashboard Cite

show abstract

“…Our results demonstrate that although both 2P and 3P excitation can be used to image through the cuticle for the superficial layers of the fly brain such as the mushroom body, 3P excitation outperforms 2P excitation in deeper brain regions such as the central complex. This conclusion is similar to that observed in the mouse brains (Mok et al, 2019;Wang et al, 2018a;Wang et al, 2020).…”

Section: Comparison Of 2p and 3p Excitation For Deep Brain Imaging Through The Fly Head Cuticlesupporting

confidence: 91%

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle

Aragon

Wang

Shea

et al. 2019

Preprint

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12A limitation of current methods for examining neural activity over long periods of time in the 13 fly, Drosophila melanogaster, is the need to remove the head cuticle and the underlying tissue 14 to gain optical access to the brain, a process that damages circulation and restricts the length 15 of imaging time. Here, we developed a non-invasive preparation for structural and functional 16 imaging of the fly brain through the intact head cuticle. We first showed that the head cuticle 17 transmits long-wavelength laser light with surprisingly high efficiencies. In fact, the tissue 18 that interferes with laser light during multiphoton imaging is the air sacs underneath the 19 head cuticle. We developed a non-invasive imaging preparation that compresses the air sacs, 20 and used it to image the mushroom body Kenyon cells and central complex ring neurons 21 through the cuticle at cellular resolution using both 2-and 3-photon microscopy. We also 22 performed non-invasive short-term and long-term functional imaging (for the first time for 23 12 consecutive hours) of odour-evoked calcium responses from the mushroom body Kenyon 24 cells. Our results demonstrate that the non-invasive imaging preparation developed here 25 extends the time limits of current in vivo imaging methods used in flies that require an 26 invasive surgery, and opens up new ways to capture neural activity from the intact fly brain.27

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“…The remote focusing strategy has also been successfully implemented in a dual-plane 2P and 3P imaging system, where 2PM and 3PM image the shallow and deep planes, respectively ( Weisenburger et al, 2019 ; Takasaki et al, 2020 ). In one work, a remote focusing module was developed to independently control the refocusing of two planes by a movable dichroic mirror and a mirror ( Mok et al, 2019 ). The beam at 920 nm for 2PM was reflected by the dichroic mirror and thus, the refocusing can be realized by shifting the dichroic mirror.…”

Section: Technical State-of-the-artmentioning

confidence: 99%

Three-photon excited fluorescence imaging in neuroscience: From principles to applications

Xiao

Deng²,

Zhao³

et al. 2023

Front. Neurosci.

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The development of three-photon microscopy (3PM) has greatly expanded the capability of imaging deep within biological tissues, enabling neuroscientists to visualize the structure and activity of neuronal populations with greater depth than two-photon imaging. In this review, we outline the history and physical principles of 3PM technology. We cover the current techniques for improving the performance of 3PM. Furthermore, we summarize the imaging applications of 3PM for various brain regions and species. Finally, we discuss the future of 3PM applications for neuroscience.

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Simultaneous Two- and Three-photon Imaging of Multilayer Neural Activities with Remote Focusing

Cited by 7 publications

References 0 publications

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle

Multiphoton imaging of neural structure and activity in Drosophila through the intact cuticle

Three-photon excited fluorescence imaging in neuroscience: From principles to applications

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