scholarly journals Fast Objective Coupled Planar Illumination Microscopy

2018 ◽  
Author(s):  
Cody Greer ◽  
Timothy E. Holy
Keyword(s):  
Fluorescence Microscopy ◽  
High Speed ◽  
Imaging Techniques ◽  
Light Sheet ◽  
Frame Rate ◽  
Three Dimensions ◽  
Two Photon Microscopy ◽  
Image Sharing ◽  
Scanning Rate ◽  
Fast Light

Among optical imaging techniques light sheet fluorescence microscopy stands out as one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. However current-generation light sheet microscopes are limited by volume scanning rate and/or camera frame rate. We present speed-optimized Objective Coupled Planar Illumination (OCPI) microscopy, a fast light sheet technique that avoids compromising image quality or photon efficiency. We increase volume scanning rate to 40 Hz for volumes up to 700 µm thick and introduce Multi-Camera Image Sharing (MCIS), a technique to scale imaging rate by parallelizing acquisition across cameras. Finally, we demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced artifact that can be removed by filtering when the imaging rate exceeds 15 Hz. These advances extend the reach of fluorescence microscopy for monitoring fast processes in large volumes.

scholarly journals Fast objective coupled planar illumination microscopy

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Cody J. Greer ◽  
Timothy E. Holy
Keyword(s):  
Fluorescence Microscopy ◽  
High Speed ◽  
Imaging Techniques ◽  
Light Sheet ◽  
Three Dimensions ◽  
Planar Imaging ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Larval Zebrafish ◽  
Fast Light

Abstract Among optical imaging techniques light sheet fluorescence microscopy is one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. However light sheet microscopes are limited by volume scanning rate and/or camera speed. We present speed-optimized Objective Coupled Planar Illumination (OCPI) microscopy, a fast light sheet technique that avoids compromising image quality or photon efficiency. Our fast scan system supports 40 Hz imaging of 700 μm-thick volumes if camera speed is sufficient. We also address the camera speed limitation by introducing Distributed Planar Imaging (DPI), a scaleable technique that parallelizes image acquisition across cameras. Finally, we demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced artifact, removable when the imaging rate exceeds 15 Hz. These advances extend the reach of fluorescence microscopy for monitoring fast processes in large volumes.

scholarly journals High-speed multifocal plane fluorescence microscopy for three-dimensional visualisation of beating flagella

2019 ◽  
Author(s):  
Richard J. Wheeler
Keyword(s):  
Fluorescence Microscopy ◽  
High Speed ◽  
Three Dimensional ◽  
Cell Movement ◽  
Frame Rate ◽  
Three Dimensions ◽  
Light Path ◽  
Leishmania Mexicana ◽  
Open Questions ◽  
Fluorescent Stain

AbstractAnalysis of flagellum beating in three dimensions is important for understanding how cells can undergo complex flagellum-driven motility and the ability to use fluorescence microscopy for such three-dimensional analysis would be extremely powerful. Trypanosoma and Leishmania are unicellular parasites which undergo complex cell movements in three dimensions as they swim and would particularly benefit from such an analysis. Here, high-speed multifocal plane fluorescence microscopy, a technique in which a light path multi-splitter is used to visualise 4 focal planes simultaneously, was used to reconstruct the flagellum beating of Trypanosoma brucei and Leishmania mexicana in three dimensions. It was possible to use either an organic fluorescent stain or a genetically-encoded fluorescence fusion protein to visualise flagellum and cell movement in three dimensions at a 200 Hz frame rate. This high-speed multifocal plane fluorescence microscopy approach was used to address two open questions regarding Trypanosoma and Leishmania swimming: To quantify the planarity of the L. mexicana flagellum beat and analyse the nature of flagellum beating during T. brucei ‘tumbling’.

scholarly journals Reflective imaging improves resolution, speed, and collection efficiency in light sheet microscopy

2017 ◽  
Author(s):  
Yicong Wu ◽  
Abhishek Kumar ◽  
Corey Smith ◽  
Evan Ardiel ◽  
Panagiotis Chandris ◽  
...  
Keyword(s):  
High Resolution ◽  
High Speed ◽  
Collection Efficiency ◽  
Single Cells ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Three Dimensions ◽  
Spatiotemporal Resolution ◽  
Light Sheet Microscopy ◽  
Simultaneous Acquisition

AbstractLight-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 μm) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

scholarly journals Implantable photonic neural probes for light-sheet fluorescence brain imaging

2020 ◽  
Author(s):  
Wesley D. Sacher ◽  
Fu-Der Chen ◽  
Homeira Moradi-Chameh ◽  
Xianshu Luo ◽  
Anton Fomenko ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Brain Imaging ◽  
Free Space ◽  
High Speed ◽  
Light Sheet ◽  
Epifluorescence Microscopy ◽  
Neural Probes ◽  
Light Sheet Fluorescence Microscopy ◽  
Brain Tissues

ABSTRACTSignificanceLight-sheet fluorescence microscopy is a powerful technique for high-speed volumetric functional imaging. However, in typical light-sheet microscopes, the illumination and collection optics impose significant constraints upon the imaging of non-transparent brain tissues. Here, we demonstrate that these constraints can be surmounted using a new class of implantable photonic neural probes.AimMass manufacturable, silicon-based light-sheet photonic neural probes can generate planar patterned illumination at arbitrary depths in brain tissues without any additional micro-optic components.ApproachWe develop implantable photonic neural probes that generate light sheets in tissue. The probes were fabricated in a photonics foundry on 200 mm diameter silicon wafers. The light sheets were characterized in fluorescein and in free space. The probe-enabled imaging approach was tested in fixed and in vitro mouse brain tissues. Imaging tests were also performed using fluorescent beads suspended in agarose.ResultsThe probes had 5 to 10 addressable sheets and average sheet thicknesses < 16 μm for propagation distances up to 300 μm in free space. Imaging areas were as large as ≈ 240 μm × 490 μm in brain tissue. Image contrast was enhanced relative to epifluorescence microscopy.ConclusionsThe neural probes can lead to new variants of light-sheet fluorescence microscopy for deep brain imaging and experiments in freely-moving animals.

scholarly journals Kilohertz frame-rate two-photon tomography

2018 ◽  
Author(s):  
Abbas Kazemipour ◽  
Ondrej Novak ◽  
Daniel Flickinger ◽  
Jonathan S. Marvin ◽  
Jonathan King ◽  
...  
Keyword(s):  
High Resolution ◽  
High Speed ◽  
Glutamate Release ◽  
Single Particle Tracking ◽  
Frame Rate ◽  
Mammalian Brain ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Calcium Sensors ◽  
Structural Image

SummaryPoint-scanning two-photon microscopy enables high-resolution imaging within scattering specimens such as the mammalian brain, but sequential acquisition of voxels fundamentally limits imaging speed. We developed a two-photon imaging technique that scans lines of excitation across a focal plane at multiple angles and uses prior information to recover high-resolution images at over 1.4 billion voxels per second. Using a structural image as a prior for recording neural activity, we imaged visually-evoked and spontaneous glutamate release across hundreds of dendritic spines in mice at depths over 250 µm and frame-rates over 1 kHz. Dendritic glutamate transients in anaesthetized mice are synchronized within spatially-contiguous domains spanning tens of microns at frequencies ranging from 1-100 Hz. We demonstrate high-speed recording of acetylcholine and calcium sensors, 3D single-particle tracking, and imaging in densely-labeled cortex. Our method surpasses limits on the speed of raster-scanned imaging imposed by fluorescence lifetime.

Investigation of Auto-Ignition of a Pulsed Methane Jet in Vitiated Air Using High-Speed Imaging Techniques

2010 ◽  
Vol 133 (2) ◽  
Author(s):  
W. Meier ◽  
I. Boxx ◽  
C. Arndt ◽  
M. Gamba ◽  
N. Clemens
Keyword(s):  
High Speed ◽  
Gas Flow ◽  
Imaging Techniques ◽  
Frame Rate ◽  
Experimental Arrangement ◽  
Single Shot ◽  
Exhaust Gas ◽  
High Speed Imaging ◽  
Auto Ignition ◽  
Flow Field Characteristics

An experimental arrangement for the investigation of auto-ignition of a pulsed CH4 jet in a coflow of hot exhaust gas from a laminar lean premixed H2/air flame at atmospheric pressure is presented. The ignition events were captured by high-speed imaging of the OH∗ chemiluminescence associated with the igniting flame kernels at a frame rate of 5 kHz. The flow-field characteristics were determined by high-speed particle image velocimetry and Schlieren images. Furthermore, high-speed imaging of laser-induced fluorescence of OH was applied to visualize the exhaust gas flow and the ignition events. Auto-ignition was observed to occur at the periphery of the CH4 jet with high reproducibility in different runs concerning time and location. In each measurement run, several hundred consecutive single shot images were recorded from which sample images are presented. The main goals of the study are the presentation of the experimental arrangement and the high-speed measuring systems and a characterization of the auto-ignition events occurring in this system.

Diffusion of Slightly Buoyant Droplets in Isotropic Turbulence

2006 ◽  
Author(s):  
Balaji Gopalan ◽  
Edwin Malkiel ◽  
Joseph Katz
Keyword(s):  
High Speed ◽  
Isotropic Turbulence ◽  
Three Dimensional ◽  
Time History ◽  
Region Of Interest ◽  
Frame Rate ◽  
Three Dimensions ◽  
Velocity Autocorrelation Function ◽  
Mean Flow ◽  
Dispersion Tensor

We study the diffusion of slightly buoyant droplets in isotropic turbulence using High Speed Digital Holographic PIV. Droplets (Specific Gravity 0.85) are injected in the central portion of an isotropic turbulence facility with weak mean flow. Perpendicular digital inline holograms are recorded in a 37 × 37 × 37 mm3 region of interest using two high speed cameras. Data are recorded at 250 frames per second (2000 frames per second is the maximum possible frame rate). An automated program is developed to obtain two dimensional tracks of the droplets from two orthogonal images and match them to get three dimensional tracks. Cross correlation of droplet images are used for measuring their velocities. The time series are low pass filtered to obtain accurate time history of droplet velocities. Data analysis determines the PDF of velocity and acceleration in three dimensions. The time history also enables us to calculate the three dimensional Lagrangian velocity autocorrelation function for different droplet radii. Integration of these functions gives us the diffusion coefficients. For shorter time scales, when the diffusion need not be Fickian we can use the three dimensional trajectories to calculate the generalized dispersion tensor and measure the time elapsed for diffusion to become Fickian.

scholarly journals Light-Sheet Microscopy in Neuroscience

2019 ◽  
Vol 42 (1) ◽  
pp. 295-313 ◽  
Author(s):  
Elizabeth M.C. Hillman ◽  
Venkatakaushik Voleti ◽  
Wenze Li ◽  
Hang Yu
Keyword(s):  
High Speed ◽  
Light Sheet ◽  
Mammalian Brain ◽  
Diverse Range ◽  
Volumetric Imaging ◽  
Two Photon Microscopy ◽  
Light Sheet Microscopy ◽  
Noise Benefits ◽  
Longitudinal Imaging

Light-sheet microscopy is an imaging approach that offers unique advantages for a diverse range of neuroscience applications. Unlike point-scanning techniques such as confocal and two-photon microscopy, light-sheet microscopes illuminate an entire plane of tissue, while imaging this plane onto a camera. Although early implementations of light sheet were optimized for longitudinal imaging of embryonic development in small specimens, emerging implementations are capable of capturing light-sheet images in freely moving, unconstrained specimens and even the intact in vivo mammalian brain. Meanwhile, the unique photobleaching and signal-to-noise benefits afforded by light-sheet microscopy's parallelized detection deliver the ability to perform volumetric imaging at much higher speeds than can be achieved using point scanning. This review describes the basic principles and evolution of light-sheet microscopy, followed by perspectives on emerging applications and opportunities for both imaging large, cleared, and expanded neural tissues and high-speed, functional imaging in vivo.

scholarly journals Capturing volumetric dynamics at high speed in the brain by confocal light field microscopy

2020 ◽  
Author(s):  
Zhenkun Zhang ◽  
Lu Bai ◽  
Lin Cong ◽  
Peng Yu ◽  
Tianlei Zhang ◽  
...  
Keyword(s):  
High Speed ◽  
Light Field ◽  
Prey Capture ◽  
Vascular System ◽  
Imaging Techniques ◽  
Three Dimensions ◽  
Proper Function ◽  
Imaging Method ◽  
Volumetric Imaging ◽  
Neural Activities

AbstractNeural network performs complex computations through coordinating collective neural dynamics that are fast and in three-dimensions. Meanwhile, its proper function relies on its 3D supporting environment, including the highly dynamic vascular system that drives energy and material flow. Better understanding of these processes requires methods to capture fast volumetric dynamics in thick tissue. This becomes challenging due to the trade-off between speed and optical sectioning capability in conventional imaging techniques. Here we present a new imaging method, confocal light field microscopy, to enable fast volumetric imaging deep into brain. We demonstrated the power of this method by recording whole brain calcium transients in freely swimming larval zebrafish and observed behaviorally correlated activities on single neurons during its prey capture. Furthermore, we captured neural activities and circulating blood cells over a volume ⌀ 800 μm × 150 μm at 70 Hz and up to 600 μm deep in the mice brain.

scholarly journals Converting lateral scanning into axial focusing to speed up three-dimensional microscopy

2020 ◽  
Vol 9 (1) ◽  
Author(s):  
Tonmoy Chakraborty ◽  
Bingying Chen ◽  
Stephan Daetwyler ◽  
Bo-Jui Chang ◽  
Oliver Vanderpoorten ◽  
...  
Keyword(s):  
High Resolution ◽  
Spherical Aberration ◽  
Optical Design ◽  
Light Sheet ◽  
Three Dimensions ◽  
High Resolution Imaging ◽  
Light Sheet Microscopy ◽  
Scanning Rate ◽  
Resolution Imaging ◽  
Axial Scanning

Abstract In optical microscopy, the slow axial scanning rate of the objective or the sample has traditionally limited the speed of volumetric imaging. Recently, by conjugating either a movable mirror to the image plane in a remote-focusing geometry or an electrically tuneable lens (ETL) to the back focal plane, rapid axial scanning has been achieved. However, mechanical actuation of a mirror limits the axial scanning rate (usually only 10–100 Hz for piezoelectric or voice coil-based actuators), while ETLs introduce spherical and higher-order aberrations that prevent high-resolution imaging. In an effort to overcome these limitations, we introduce a novel optical design that transforms a lateral-scan motion into a spherical aberration-free axial scan that can be used for high-resolution imaging. Using a galvanometric mirror, we scan a laser beam laterally in a remote-focusing arm, which is then back-reflected from different heights of a mirror in the image space. We characterize the optical performance of this remote-focusing technique and use it to accelerate axially swept light-sheet microscopy by an order of magnitude, allowing the quantification of rapid vesicular dynamics in three dimensions. We also demonstrate resonant remote focusing at 12 kHz with a two-photon raster-scanning microscope, which allows rapid imaging of brain tissues and zebrafish cardiac dynamics with diffraction-limited resolution.

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