Video-rate volumetric neuronal imaging using 3D targeted illumination

Xiao, Sheng; Tseng, Hua-an; Gritton, Howard J.; Han, Xue; Mertz, Jérôme

doi:10.1038/s41598-018-26240-8

Cited by 25 publications

(19 citation statements)

References 41 publications

(43 reference statements)

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“…In contrast, relatively little absorption and much less scattering occurs in the so-called tissue transparency window from ~700–900 nm [76]. Thus, effective optogenetic monitoring and control of neurons and other cells in tissues is severely limited to depths of only a few mm for most in vivo applications without using NIR wavelengths and advanced NIR optical techniques which can take advantage of this transparency window [76–79]. In this regard, the development of NIR excitable and emitting microbial rhodopsin voltage sensors and membrane voltage modulators would be particularly attractive for monitoring or manipulating electrical activity in the brain, especially to simultaneously measure the activity of millions of individual neurons over large volumes of tissue.…”

Section: Discussionmentioning

confidence: 99%

Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate

et al. 2018

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Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified β-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH <7.3, and the visible bands dominant at pH 9.5. FT-Raman using 1064-nm excitation reveal two strong ethylenic bands at 1482 and 1498 cm-1 corresponding to the NIR subcomponent absorption bands based on an extended linear correlation between λmax and γC = C. This spectrum exhibits two intense bands in the fingerprint and HOOP mode regions that are highly characteristic of the O640 photointermediate from the light-adapted bacteriorhodopsin photocycle. In contrast, 532-nm excitation enhances the 560-nm component, which exhibits bands very similar to light-adapted bacteriorhodopsin and/or the acid-purple form of bacteriorhodopsin. Native GPR and its mutant D97N when regenerated with MMAR also exhibit similar absorption and Raman bands but with weaker contributions from the NIR absorbing components. Based on these results it is proposed that the NIR absorption in GPR-D212N/F234S with MMAR arises from an O-like chromophore, where the Schiff base counterion D97 is protonated and the MMAR adopts an all-trans configuration with a non-planar geometry due to twists in the conjugated polyene segment. This configuration is characterized by extensive charge delocalization, most likely involving nitrogens atoms in the MMAR chromophore.

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Section: Discussionmentioning

confidence: 99%

Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate

et al. 2018

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“…Spatial overlap is profound for wide-field imaging, but not for two-photon imaging or in vitro cell culture imaging with single cell layer. With increasing improvement wide-field imaging, such as volumetric imaging (Shain et al, 2018; Xiao et al, 2018), such significant overlap may be better eliminated during data acquisition step.…”

Section: Discussionmentioning

confidence: 99%

Automatic Cell Segmentation by Adaptive Thresholding (ACSAT) for Large-Scale Calcium Imaging Datasets

Shen

Tseng

Hansen

et al. 2018

eNeuro

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Advances in calcium imaging have made it possible to record from an increasingly larger number of neurons simultaneously. Neuroscientists can now routinely image hundreds to thousands of individual neurons. An emerging technical challenge that parallels the advancement in imaging a large number of individual neurons is the processing of correspondingly large datasets. One important step is the identification of individual neurons. Traditional methods rely mainly on manual or semimanual inspection, which cannot be scaled for processing large datasets. To address this challenge, we focused on developing an automated segmentation method, which we refer to as automated cell segmentation by adaptive thresholding (ACSAT). ACSAT works with a time-collapsed image and includes an iterative procedure that automatically calculates global and local threshold values during successive iterations based on the distribution of image pixel intensities. Thus, the algorithm is capable of handling variations in morphological details and in fluorescence intensities in different calcium imaging datasets. In this paper, we demonstrate the utility of ACSAT by testing it on 500 simulated datasets, two wide-field hippocampus datasets, a wide-field striatum dataset, a wide-field cell culture dataset, and a two-photon hippocampus dataset. For the simulated datasets with truth, ACSAT achieved >80% recall and precision when the signal-to-noise ratio was no less than ∼24 dB.

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“…Parallel to the molecular targeting of GEVIs, targeted illumination has also been developed to enhance image contrast and signal-to-noise ratio (SNR) ( Adam et al., 2019 ; Fan et al., 2020 ; Xiao et al., 2018 ). By using a digital micromirror device (DMD) or a spatial light modulator to pattern the excitation light, targeted illumination confines fluorescent excitation to preselected areas of interest.…”

Section: Introductionmentioning

confidence: 99%

Large-scale voltage imaging in behaving mice using targeted illumination

et al. 2021

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Summary Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold transmembrane voltage in vivo . To perform high-speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes widefield microscopy prone to background cross-contamination. We implemented a digital-micromirror-device-based targeted illumination strategy to restrict illumination to the cells of interest and quantified the resulting improvement both theoretically and experimentally with SomArchon expressing neurons. We found that targeted illumination increased SomArchon signal contrast, decreased photobleaching, and reduced background cross-contamination. With the use of a high-speed, large-area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in behaving mice. Thus, the targeted illumination strategy described here offers a simple solution for widefield voltage imaging of many neurons over a large field of view in behaving animals.

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Video-rate volumetric neuronal imaging using 3D targeted illumination

Cited by 25 publications

References 41 publications

Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate

Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate

Automatic Cell Segmentation by Adaptive Thresholding (ACSAT) for Large-Scale Calcium Imaging Datasets

Large-scale voltage imaging in behaving mice using targeted illumination

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