Video-rate volumetric functional imaging of the brain at synaptic resolution

Lü, Rong; Sun, Wenzhi; Liang, Yajie; Kerlin, Aaron; Bierfeld, Jens; Seelig, Johannes D.; Wilson, Dana E.; Scholl, Benjamin; Mohar, Boaz; Tanimoto, Masashi; Koyama, Minoru; Fitzpatrick, David; Orger, Michael B.; Ji, Na

doi:10.1038/nn.4516

Cited by 255 publications

(200 citation statements)

References 60 publications

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“…The most common approach involves the use of an axicon (conical lens) [8, 9, 10]. Other approaches employed a phase mask [11], or alternatively a spatial light modulator (SLM) [12], with the latter allowing for great flexibility in generating different types of Bessel foci with shaped axial intensity profiles.…”

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confidence: 99%

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Three-photon fluorescence microscopy with an axially elongated Bessel focus

Rodríguez

Liang

et al. 2018

Opt. Lett.

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Volumetric imaging tools that are simple to adopt, flexible, and robust, are in high demand in the field of neuroscience, where the ability to image neurons and their networks with high spatiotemporal resolution is essential. Using an axially elongated focus approximating a Bessel beam, in combination with two-photon fluorescence microscopy, has proven successful at such an endeavor. Here we demonstrate three-photon fluorescence imaging with an axially extended Bessel focus. We use an axicon-based module which allowed for the generation of Bessel foci of varying numerical aperture and axial length, and apply this volumetric imaging tool to image mouse brain slices and for in vivo imaging of the mouse brain.

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confidence: 99%

“…Previous observations showed that, for two-photon fluorescence microscopy, lower-NA (e.g., 0.4 or 0.6) Bessel foci produced better-quality images of brains in vivo [12]. For high-NA Bessel foci (e.g., 0.9), more energy is distributed in the side rings, causing a stronger background and image blur.…”

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confidence: 99%

Three-photon fluorescence microscopy with an axially elongated Bessel focus

Rodríguez

Liang

et al. 2018

Opt. Lett.

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“…In other words, the volumetric information is projected into a single 2D image. As such, extended depth of focus (EDF) imaging can be attractive to image sparsely populated structures rapidly and has found promising applications in functional imaging of neuronal activity [1,2].…”

Section: Introductionmentioning

confidence: 99%

“…Various beam shaping methods have been successfully employed for EDF imaging, which involve different amplitude, phase or combined filters in the pupil plane [3][4][5], and in some cases also feature specialized optical elements such as axicons [6,7]. Their implementation typically requires significant changes to a conventional microscope [1]. Further, if phase modulation is used, the associated phase masks or holograms are typically wavelength dependent, which prevents, or at least complicates, multi-color imaging.…”

Section: Introductionmentioning

confidence: 99%

Extended depth of focus multiphoton microscopy via incoherent pulse splitting

Chen

Chakraborty

Daetwyler

et al. 2020

Preprint

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We present a phase mask that can be easily added to any multi-photon raster scanning microscope to extend the depth of focus five-fold at a small loss in lateral resolution. The method is designed for ultrafast laser pulses or other light-sources featuring a low coherence length. In contrast to other methods of focus extension, our approach uniquely combines low complexity, high light-throughput and multicolor capability. We characterize the point-spread function in a two-photon microscope and demonstrate fluorescence imaging of GFP labeled neurons in fixed brain samples as imaged with conventional and extended depth of focus two-photon microscopy.

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“…Investigating the operation of these circuits has been greatly advanced by using multiphoton microscopy to image fluorescent reporter proteins that signal events such as changes in cytoplasmic calcium, reflecting spiking activity (Tian et al, 2012), or the release of glutamate at excitatory synapses (Marvin et al, 2013). At present, the large majority of in vivo imaging studies quantify activity as calcium signals in the soma of neurons but we also need to image activity across populations of synapses if the operations of neural circuits are to be unravelled (Lu et al, 2017;Kazemipour et al, 2019;Meng et al, 2019). These connections are key components of all the computations within neural circuits and sites at which neuromodulators act to reconfigure signal flow according to changes in the internal state of the animal.…”

Section: Introductionmentioning

confidence: 99%

Correction of z-motion artefacts to allow population imaging of synaptic activity in awake behaving mice

Ryan

Hinojosa

Vroman

et al. 2019

Preprint

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Motion artefacts associated with motor behaviour are an inevitable problem of multiphoton imaging in awake behaving animals, particularly imaging synapses.• Correction of axial motion usually requires volumetric imaging resulting in slower rates of acquisition.• We describe a method that is easy to implement to correct z-motion artefacts that allows population imaging of synaptic activity while scanning a single plane in a standard multiphoton microscope.• The method uses a reference volume acquired in two colour channels -an activity reporter and an anatomical marker of blood vessels. The procedure estimates the zdisplacement in every frame and applies an intensity correction in which the z pointspread function for each synapse is modelled as a Moffat function.• We demonstrate that the method allows synaptic calcium activity signals to be collected from populations of synaptic boutons in mouse primary visual cortex during locomotion. AbstractFunctional imaging of head-fixed, awake, behaving mice using two-photon imaging of fluorescent activity reporters has become a powerful tool in the studying the function of the brain. Motion artefacts are an inevitable problem during such experiments and are routinely corrected for in x and y dimensions. However, axial (z) shifts of several microns can also occur, leading to intensity fluctuations in structures such as synapses that are small compared to the axial point-spread function of the microscope. Here we present a simple strategy to correct z-motion artefacts arising over the course of a time-series experiment in a single optical plane. Displacement in z was calculated using dye-filled blood vessels as an anatomical marker, providing high contrast images and accuracy to within ~0.1 µm. The axial profiles of ROIs corresponding to synapses were described using a Moffat function and this "ROI-spread function" used to correct activity traces on an ROIby-ROI basis. We demonstrate the accuracy and utility of the procedures in simulation experiments using fluorescent beads and then apply them to correcting measurements of synaptic activity in populations of vasoactive-intestinal peptide (VIP) interneurons expressing the synaptic reporter SyGCaMP6f. Correction of z-motion artefacts had a substantial impact on the apparent correlation between synaptic activity and running speed, demonstrating the importance of correcting for these artefacts for the interpretation of in vivo imaging experiments in awake mice.

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Video-rate volumetric functional imaging of the brain at synaptic resolution

Cited by 255 publications

References 60 publications

Three-photon fluorescence microscopy with an axially elongated Bessel focus

Three-photon fluorescence microscopy with an axially elongated Bessel focus

Extended depth of focus multiphoton microscopy via incoherent pulse splitting

Correction of z-motion artefacts to allow population imaging of synaptic activity in awake behaving mice

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