Video-rate large-scale imaging with Multi-Z confocal microscopy

Badon, Amaury; Bensussen, Seth; Gritton, Howard J.; Awal, Mehraj R.; Gabel, Christopher V.; Han, Xue; Mertz, Jérôme

doi:10.1364/optica.6.000389

Cited by 41 publications

(30 citation statements)

References 37 publications

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“…Imaging was performed using a custom-built confocal microscope at a frame rate of 458 Hz using a 16X/0.8 NA water-immersion objective lens (Nikon CFI75 LWD 16X W). High frame rates were achieved using a system similar to that described previously (Badon et al 2019) but with a 128-facet polygonal scanner (Cambridge Technology SA34) substituted for the x-axis scanner. Voltron2585 was excited with a 561 nm laser diode (Vortran Stradus).…”

Section: Simultaneous Voltage Imaging and Optogenetic Stimulation In Brain Slicesmentioning

confidence: 99%

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

Abdelfattah

Zheng

Reep

et al. 2021

Preprint

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The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased ΔF/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

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Section: Simultaneous Voltage Imaging and Optogenetic Stimulation In Brain Slicesmentioning

confidence: 99%

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

Abdelfattah

Zheng

Reep

et al. 2021

Preprint

Self Cite

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“…Although these modalities can achieve 3D images at relatively high resolution, the axial scanning rate limits the speed of volumetric imaging. Although alternative strategies including confocal microscopy with multiple pinholes/slits/prisms [12][13][14] which enables volumetric imaging at a video rate in a relatively large field of view, these methodologies suffer from a limited number of layers detected in the axial direction as well as relatively low optical resolution.…”

Section: Introductionmentioning

confidence: 99%

Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

Mac

Qureshi

et al. 2021

Preprint

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In microscopic imaging of biological tissues, particularly real-time visualization of neuronal activities, rapid acquisition of volumetric images poses a prominent challenge. Typically, two-dimensional (2D) microscopy can be devised into an imaging system with 3D capability using any varifocal lens. Despite the conceptual simplicity, such an upgrade yet requires additional, complicated device components and suffers a reduced acquisition rate, which is critical to document neuronal dynamics properly. In this study, we implemented an electro-tunable lens (ETL) in the line-scan confocal microscopy, enabling the volumetric acquisition at the rate of 20 frames per second with the maximum volume of interest of 315 × 315 × 80 μm3. The axial extent of point-spread-function (PSF) was 17.6 ± 1.6 μm and 90.4 ± 2.1 μm with the ETL operating in either stationary or resonant mode, respectively, revealing significant depth elongation by the resonant mode ETL microscopy. We further demonstrated the utilities of the ETL system by volume imaging of cleared mouse brain ex vivo samples and in vivo brains. The current study foregrounds the successful application of resonant ETL for constructing a basis for a high-performance 3D line-scan confocal microscopy system, which will enhance our understanding of various dynamic biological processes.

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“…This is a critical requirement that ensures high-degree of viability of a biological sample. However, existing techniques of parallelization are either restricted to two dimensions (e.g., LSFM 3,7,8 ) or sparse sampling in three dimensions (c.f., multi-focal [9][10][11] or multi-light-sheet imaging [12][13][14][15][16][17][18] ). In particular, the current multi-light-sheet fluorescence imaging systems, including the recent advances in lattice light-sheet microscopy, commonly resort to coherent beam interference, beam split, or wavefront shaping (in the spatial or Fourier domain) [12][13][14][15]19 .…”

Section: Introductionmentioning

confidence: 99%

Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array

Ren

Lai

et al. 2020

Light Sci Appl

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Parallelized fluorescence imaging has been a long-standing pursuit that can address the unmet need for a comprehensive three-dimensional (3D) visualization of dynamical biological processes with minimal photodamage. However, the available approaches are limited to incomplete parallelization in only two dimensions or sparse sampling in three dimensions. We hereby develop a novel fluorescence imaging approach, called coded light-sheet array microscopy (CLAM), which allows complete parallelized 3D imaging without mechanical scanning. Harnessing the concept of an "infinity mirror", CLAM generates a light-sheet array with controllable sheet density and degree of coherence. Thus, CLAM circumvents the common complications of multiple coherent light-sheet generation in terms of dedicated wavefront engineering and mechanical dithering/scanning. Moreover, the encoding of multiplexed optical sections in CLAM allows the synchronous capture of all sectioned images within the imaged volume. We demonstrate the utility of CLAM in different imaging scenarios, including a light-scattering medium, an optically cleared tissue, and microparticles in fluidic flow. CLAM can maximize the signal-to-noise ratio and the spatial duty cycle, and also provides a further reduction in photobleaching compared to the major scanning-based 3D imaging systems. The flexible implementation of CLAM regarding both hardware and software ensures compatibility with any light-sheet imaging modality and could thus be instrumental in a multitude of areas in biological research.

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Video-rate large-scale imaging with Multi-Z confocal microscopy

Cited by 41 publications

References 37 publications

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

Fast volumetric imaging with line-scan confocal microscopy by an electro-tunable lens

Parallelized volumetric fluorescence microscopy with a reconfigurable coded incoherent light-sheet array

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