Video-rate Volumetric Neuronal Imaging Using 3D Targeted Illumination

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

doi:10.1364/brain.2018.bw2c.6

Cited by 4 publications

(4 citation statements)

References 28 publications

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“…To overcome the intrinsic optical constraints of conventional fluorescence microscopy for extended DOF, different approaches have been employed, such as decoupled illumination and detection in light-sheet microscopy (7), dynamic remote focusing (8,9), and spatial and spectral multiplexing (10,11); nonetheless, they usually require customized and expensive optics or complicated geometrical configurations. Alternatively, reflectance-based label-free modalities, including reflectance confocal microscopy and full-field optical coherence tomography, have been demonstrated for cancer-lesion characterization in skin and different types of epithelium (12)(13)(14)(15).…”

Section: Significancementioning

confidence: 99%

Deep learning extended depth-of-field microscope for fast and slide-free histology

Jin

Tang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Microscopic evaluation of resected tissue plays a central role in the surgical management of cancer. Because optical microscopes have a limited depth-of-field (DOF), resected tissue is either frozen or preserved with chemical fixatives, sliced into thin sections placed on microscope slides, stained, and imaged to determine whether surgical margins are free of tumor cells—a costly and time- and labor-intensive procedure. Here, we introduce a deep-learning extended DOF (DeepDOF) microscope to quickly image large areas of freshly resected tissue to provide histologic-quality images of surgical margins without physical sectioning. The DeepDOF microscope consists of a conventional fluorescence microscope with the simple addition of an inexpensive (less than $10) phase mask inserted in the pupil plane to encode the light field and enhance the depth-invariance of the point-spread function. When used with a jointly optimized image-reconstruction algorithm, diffraction-limited optical performance to resolve subcellular features can be maintained while significantly extending the DOF (200 µm). Data from resected oral surgical specimens show that the DeepDOF microscope can consistently visualize nuclear morphology and other important diagnostic features across highly irregular resected tissue surfaces without serial refocusing. With the capability to quickly scan intact samples with subcellular detail, the DeepDOF microscope can improve tissue sampling during intraoperative tumor-margin assessment, while offering an affordable tool to provide histological information from resected tissue specimens in resource-limited settings.

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Section: Significancementioning

confidence: 99%

Deep learning extended depth-of-field microscope for fast and slide-free histology

Jin

Tang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Several techniques have been proposed to expand the DOF. Reducing the numerical aperture (NA) increases the depth of field, but leads to low resolution and low signal to noise ratio (SNR), inducing photo-bleaching and phototoxify; Fast focal distance adjustment elements, such as electrically tunable lens and TAG lens, encounter the tradeoff among FOV, resolution and speed, which are thus inapplicable to high throughput lens [9][10][11][12] ; Piezo stages under resonance frequency cannot achieve fast scan and high load simultaneously, which is also challenging to achieve accurate synchronization 13 ; focal length expending techniques, such as SPED microscopy and cubic phase plate-base techniques, acquire larger field of view at the cost of lower SNR [14][15][16] ; 3D imaging techniques, such as light field microscopy [17][18][19][20][21] , sacrifice the resolution for improved speed, but bring high computation cost. Besides, all of these approaches improve imaging speed but suffer from optical artifacts (especially beyond the native focal plane or point).…”

Section: Introductionmentioning

confidence: 99%

Spinning Disk Multifocal Microscopy for Dynamic Arbitrarily Shaped Surface Imaging at Centimetre Scale and Micrometre Resolution

Xie

Han

Xiao

et al. 2022

Preprint

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The large-scale fluorescence microscopy has enabled the observation of dynamic physiological activities at the single cellular level across the mouse cortex, such as distributed neuronal population representations. However, video-rate high-resolution microscopy at sophisticated biological surfaces in nature keeps a challenging task for the tradeoff between the speed, resolution, and field of view. Here we propose Spinning Disk Multifocal Microscopy (SDiM) for arbitrarily shaped surfaces, which enables imaging at centimeter field-of-view, micrometer resolution and up to 30 frames per second across the depth range of 450 μm. We apply this technique in various microscopic systems, including customized macroscopic systems and the Real-time Ultra-large-Scale imaging at High resolution macroscopy (RUSH), in both the reflective mode and the fluorescence mode, and in the study of cortex-wide single-neuron imaging and immune cell tracking. SDiM provides an opportunity for studying the cortex-wide cellular interactions.

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“…In parallel with molecular targeting of GEVIs, targeted illumination in optical microscopy design has been proposed as a simple strategy to improve upon a widefield microscopy for enhancing image contrast and improving signal-to-noise ratio (SNR) 23 . For example, by targeting illumination to cell bodies or plasma membranes, voltage imaging performance has been significantly improved [4][5][6] .…”

Section: Introductionmentioning

confidence: 99%

Large-scale voltage imaging in the brain using targeted illumination

Xiao

Lowet

Gritton

et al. 2021

Preprint

Self Cite

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Recent improvements in genetically encoded voltage indicators enabled high precision imaging of single neuron's action potentials and subthreshold membrane voltage dynamics in the mammalian brain. To perform high speed voltage imaging, widefield microscopy remains an essential tool to record activity from many neurons simultaneously over a large anatomical area. However, the lack of optical sectioning makes widefield microscopy prone to background signal contamination. We implemented a simple, low cost, targeted illumination strategy based on a digital micromirror device (DMD) to restrict illumination to the cells of interest to improve background rejection, and quantified optical voltage signal improvement in neurons expressing the fully genetically encoded voltage indicator SomArchon. We found that targeted illumination, in comparison to widefield illumination, increased SomArchon signal contrast and reduced background cross-contamination in the brains of awake mice. Such improvement permitted the reduction of illumination intensity, and thus reduced fluorescence photobleaching and prolonged imaging duration. When coupled with a high-speed sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over several minutes in the brain. Thus, the DMD-based targeted illumination strategy described here offers a simple solution for high-speed voltage imaging analysis of large scale network at the millisecond time scale with single cell resolution in the brains of behaving animals.

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Video-rate Volumetric Neuronal Imaging Using 3D Targeted Illumination

Cited by 4 publications

References 28 publications

Deep learning extended depth-of-field microscope for fast and slide-free histology

Deep learning extended depth-of-field microscope for fast and slide-free histology

Spinning Disk Multifocal Microscopy for Dynamic Arbitrarily Shaped Surface Imaging at Centimetre Scale and Micrometre Resolution

Large-scale voltage imaging in the brain using targeted illumination

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