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 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.

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.

Confocal fluorescence microscopy with the tandem scanning light microscope

1989 ◽  
Vol 94 (4) ◽  
pp. 617-624
Author(s):  
S.J. Wright ◽  
J.S. Walker ◽  
H. Schatten ◽  
C. Simerly ◽  
J.J. McCarthy ◽  
...  
Keyword(s):  
High Resolution ◽  
Fluorescence Microscopy ◽  
Light Microscope ◽  
Three Dimensional ◽  
Three Dimensions ◽  
Complex Structures ◽  
Charge Coupled Device ◽  
Confocal Fluorescence ◽  
Cooled Ccd ◽  
Cellular Components

Applications of the tandem scanning confocal microscope (TSM) to fluorescence microscopy and its ability to resolve fluorescent biological structures are described. The TSM, in conjunction with a cooled charge-coupled device (cooled CCD) and conventional epifluorescence light source and filter sets, provided high-resolution, confocal data, so that different fluorescent cellular components were distinguished in three dimensions within the same cell. One of the unique features of the TSM is the ability to image fluorochromes excited by ultraviolet light (e.g. Hoechst, DAPI) in addition to fluorescein and rhodamine. Since the illumination is dim, photobleaching is insignificant and prolonged viewing of living specimens is possible. Series of optical sections taken in the Z-axis with the TSM were reproduced as stereo images and three-dimensional reconstructions. These data show that the TSM is potentially a powerful tool in fluorescence microscopy for determining three-dimensional relationships of complex structures within cells labeled with multiple fluorochromes.

Three-dimensional reconstruction of metabolic data from quantitative autoradiography of rat brain

1984 ◽  
Vol 247 (3) ◽  
pp. E412-E419 ◽  
Author(s):  
L. S. Hibbard ◽  
R. A. Hawkins
Keyword(s):  
High Speed ◽  
Three Dimensional ◽  
Image Data ◽  
Brain Structures ◽  
Three Dimensions ◽  
Quantitative Autoradiography ◽  
Alignment Algorithm ◽  
Computer Methods ◽  
Mathematical Properties ◽  
Dimensional Reconstruction

Quantitative autoradiography is a powerful method for studying brain function by the determination of blood flow, glucose utilization, or transport of essential nutrients. Autoradiographic images contain vast amounts of potentially useful information, but conventional analyses can practically sample the data at only a small number of points arbitrarily chosen by the experimenter to represent discrete brain structures. To use image data more fully, computer methods for its acquisition, storage, quantitative analysis, and display are required. We have developed a system of computer programs that performs these tasks and has the following features: 1) editing and analysis of single images using interactive graphics, 2) an automatic image alignment algorithm that places images in register with one another using only the mathematical properties of the images themselves, 3) the calculation of mean images from equivalent images in different experimental serial image sets, 4) the calculation of difference images (e.g., experiment-minus-control) with the option to display only differences estimated to be statistically significant, and 5) the display of serial image metabolic maps reconstructed in three dimensions using a high-speed computer graphics system.

Three Dimensional Imaging of LIGA-Made Microcomponents

2004 ◽  
Vol 126 (4) ◽  
pp. 813-821 ◽  
Author(s):  
Douglas Chinn ◽  
Peter Ostendorp ◽  
Mike Haugh ◽  
Russell Kershmann ◽  
Thomas Kurfess ◽  
...  
Keyword(s):  
Computer Aided Design ◽  
High Speed ◽  
Three Dimensional ◽  
Point Clouds ◽  
Three Dimensions ◽  
Cad Model ◽  
Volumetric Imaging ◽  
Data Registration ◽  
Liga Technology ◽  
Coordinate Metrology

Nickel and nickel-alloy microparts sized on the order of 5–1000 microns have been imaged in three dimensions using a new microscopic technique, Digital Volumetric Imaging (DVI). The gears were fabricated using Sandia National Laboratories’ LIGA technology (lithography, molding, and electroplating). The images were taken on a microscope built by Resolution Sciences Corporation by slicing the gear into one-micron thin slices, photographing each slice, and then reconstructing the image with software. The images were matched to the original CAD (computer aided design) model, allowing LIGA designers, for the first time, to see visually how much deviation from the design is induced by the manufacturing process. Calibration was done by imaging brass ball bearings and matching them to the CAD model of a sphere. A major advantage of DVI over scanning techniques is that internal defects can be imaged to very high resolution. In order to perform the metrology operations on the microcomponents, high-speed and high-precision algorithms are developed for coordinate metrology. The algorithms are based on a least-squares approach to data registration the {X,Y,Z} point clouds generated from the component surface onto a target geometry defined in a CAD model. Both primitive geometric element analyses as well as an overall comparison of the part geometry are discussed. Initial results of the micromeasurements are presented in the paper.

Blink-Spot Projection Method for Fast Three-Dimensional Shape Measurement

2015 ◽  
Vol 27 (4) ◽  
pp. 430-443 ◽  
Author(s):  
Jun Chen ◽  
◽  
Qingyi Gu ◽  
Tadayoshi Aoyama ◽  
Takeshi Takaki ◽  
...  
Keyword(s):  
Projection Method ◽  
Fast Moving ◽  
High Speed ◽  
Degrees Of Freedom ◽  
Moving Objects ◽  
Three Dimensional ◽  
Frame Rate ◽  
Light Illumination ◽  
Camera System ◽  
3D Shape

<div class=""abs_img""> <img src=""[disp_template_path]/JRM/abst-image/00270004/13.jpg"" width=""300"" /> Blink-spot projection method</div> We present a blink-spot projection method for observing moving three-dimensional (3D) scenes. The proposed method can reduce the synchronization errors of the sequential structured light illumination, which are caused by multiple light patterns projected with different timings when fast-moving objects are observed. In our method, a series of spot array patterns, whose spot sizes change at different timings corresponding to their identification (ID) number, is projected onto scenes to be measured by a high-speed projector. Based on simultaneous and robust frame-to-frame tracking of the projected spots using their ID numbers, the 3D shape of the measuring scene can be obtained without misalignments, even when there are fast movements in the camera view. We implemented our method with a high-frame-rate projector-camera system that can process 512 × 512 pixel images in real-time at 500 fps to track and recognize 16 × 16 spots in the images. Its effectiveness was demonstrated through several 3D shape measurements when the 3D module was mounted on a fast-moving six-degrees-of-freedom manipulator. </span>

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 A FRAMEWORK FOR RELIABLE THREE-DIMENSIONAL UNDERGROUND UTILITY MAPPING FOR URBAN PLANNING

2018 ◽  
Vol XLII-4/W10 ◽  
pp. 209-214 ◽  
Author(s):  
R. van Son ◽  
S. W. Jaw ◽  
J. Yan ◽  
V. Khoo ◽  
R. Loo ◽  
...  
Keyword(s):  
Urban Planning ◽  
High Speed ◽  
Large Scale ◽  
Three Dimensional ◽  
Research Literature ◽  
Three Dimensions ◽  
3D Mapping ◽  
Literature Study ◽  
Underground Utilities ◽  
Utility Services

<p><strong>Abstract.</strong> To optimise the use of limited available land, land-scarce cities such as Singapore are increasingly looking towards the underground in search of more space. A good understanding of what already exists underground is essential for the planning of underground spaces. In particular, utility services make up a significant part of what exists underground. To meet planning needs, the Singapore government has initiated efforts towards bringing records of existing utility networks together in a single database and share its contents to support planning, design, and construction of underground developments. However, these records can not be relied on to support these critical processes: They are not guaranteed to represent today’s state of the underground, are not accurate or of unknown accuracy, are inconsistently modelled, and may indicate as-design information instead of as-built information. This lack of reliability leads to an increase in cost and a loss in efficiency caused by the need to repeatedly survey to locate existing utility services on-site, and can have potentially disastrous outcomes when an excavation would damage existing services. Technological advances in utility surveying and mapping devices such as Ground Penetrating Radar (GPR) and gyroscopic pipeline mapping devices offer the potential of accurately mapping utilities in three dimensions (3D) at a large scale and high speed. However, a better understanding of the benefits and limitations of these technologies in a practical context is needed, as well as their suitability for mapping to support applications such as urban planning and land administration. The Digital Underground project is a collaboration between Singapore-ETH Centre, Singapore Land Authority and the City of Zürich that aims to develop a roadmap towards a reliable 3D utility map of Singapore. To enable the development of utility mapping standards and guidelines, the 3D mapping workflow for underground utilities is studied extensively based on market research, literature study, and case studies. This work presents the beginnings of a framework for 3D mapping of underground utilities as one of the initial results of the Digital Underground project as it is in progress. From these experiences, it can be concluded that, together with existing data, data captured using various surveying methods can indeed contribute to the establishment and maintenance of a consolidated and reliable utility map. To this end, a multi-sensor, multi-data 3D mapping workflow is proposed to integrate data captured using different surveying techniques during different moments in the development lifecycle of utilities. Based on this framework, this work also identifies areas for improvement and critical gaps to be bridged that will ultimately form part of the roadmap.</p>

High Speed Micro Holographic PIV Measurements of Microorganisms

2008 ◽  
Author(s):  
M. Zarzecki ◽  
F. J. Diez
Keyword(s):  
Velocity Field ◽  
High Speed ◽  
Fourier Transforms ◽  
Three Dimensional ◽  
Three Dimensions ◽  
Imaging Method ◽  
Time Resolved ◽  
Feeding Methods ◽  
Holographic Particle Image Velocimetry ◽  
Three Components

Holographic particle image velocimetry (PIV) is a novel application of holography that allows for tracking of small particle sized objects in a small volume. Whereas regular PIV allows for the two in-plane components of the velocity field to be measured, and stereoscopic PIV allows for the three-components of the velocity field to be measured in a thin plane, holographic PIV allows for the three-components of the velocity to be measured for each individual particle present in the measuring volume, thus allowing to fully resolve fluid flows that are inherently 3D in nature. There are many examples of three dimensional flows in nature including turbulence flows, but another very interesting application very well suited for this technique involves tracking living microorganisms in order to study their motion and their means of propulsion. As part of this research a micro organism was tracked in three dimensions using a high speed microscopic holographic imaging method. The ability to track organisms in 3D allows better understanding and characterizing of their behavior including their propulsion methods, their feeding methods and their interaction with each other. The time resolved holograms were reconstructed in Matlab using Fast Fourier Transforms. A laser pointer was used as a source of coherent light, and a high speed PIV camera (Photron APX Ultima) was used to capture the images. A beam expander was used to increase the diameter of the laser beam allowing for a larger tracking area. Results with this system will show the trajectories in 3D of microorganisms as well as the three components of the velocity field showing the interaction of the organisms with their environment.

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