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

2019 ◽  
Vol 132 (16) ◽  
pp. jcs231795 ◽  
Author(s):  
Benjamin J. Walker ◽  
Richard J. Wheeler
2019 ◽  
Author(s):  
Richard J. Wheeler

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’.


Author(s):  
Brian Cross

A relatively new entry, in the field of microscopy, is the Scanning X-Ray Fluorescence Microscope (SXRFM). Using this type of instrument (e.g. Kevex Omicron X-ray Microprobe), one can obtain multiple elemental x-ray images, from the analysis of materials which show heterogeneity. The SXRFM obtains images by collimating an x-ray beam (e.g. 100 μm diameter), and then scanning the sample with a high-speed x-y stage. To speed up the image acquisition, data is acquired "on-the-fly" by slew-scanning the stage along the x-axis, like a TV or SEM scan. To reduce the overhead from "fly-back," the images can be acquired by bi-directional scanning of the x-axis. This results in very little overhead with the re-positioning of the sample stage. The image acquisition rate is dominated by the x-ray acquisition rate. Therefore, the total x-ray image acquisition rate, using the SXRFM, is very comparable to an SEM. Although the x-ray spatial resolution of the SXRFM is worse than an SEM (say 100 vs. 2 μm), there are several other advantages.


Author(s):  
Robert W. Mackin

This paper presents two advances towards the automated three-dimensional (3-D) analysis of thick and heavily-overlapped regions in cytological preparations such as cervical/vaginal smears. First, a high speed 3-D brightfield microscope has been developed, allowing the acquisition of image data at speeds approaching 30 optical slices per second. Second, algorithms have been developed to detect and segment nuclei in spite of the extremely high image variability and low contrast typical of such regions. The analysis of such regions is inherently a 3-D problem that cannot be solved reliably with conventional 2-D imaging and image analysis methods.High-Speed 3-D imaging of the specimen is accomplished by moving the specimen axially relative to the objective lens of a standard microscope (Zeiss) at a speed of 30 steps per second, where the stepsize is adjustable from 0.2 - 5μm. The specimen is mounted on a computer-controlled, piezoelectric microstage (Burleigh PZS-100, 68/μm displacement). At each step, an optical slice is acquired using a CCD camera (SONY XC-11/71 IP, Dalsa CA-D1-0256, and CA-D2-0512 have been used) connected to a 4-node array processor system based on the Intel i860 chip.


2021 ◽  
Author(s):  
Scott J. Peltier ◽  
Brian E. Rice ◽  
Ethan Johnson ◽  
Venkateswaran Narayanaswamy ◽  
Marvin E. Sellers

2018 ◽  
Author(s):  
Yi Chen Mazumdar ◽  
Michael E. Smyser ◽  
Jeffery Dean Heyborne ◽  
Daniel Robert Guildenbecher

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiang Lan Fan ◽  
Jose A. Rivera ◽  
Wei Sun ◽  
John Peterson ◽  
Henry Haeberle ◽  
...  

AbstractUnderstanding the structure and function of vasculature in the brain requires us to monitor distributed hemodynamics at high spatial and temporal resolution in three-dimensional (3D) volumes in vivo. Currently, a volumetric vasculature imaging method with sub-capillary spatial resolution and blood flow-resolving speed is lacking. Here, using two-photon laser scanning microscopy (TPLSM) with an axially extended Bessel focus, we capture volumetric hemodynamics in the awake mouse brain at a spatiotemporal resolution sufficient for measuring capillary size and blood flow. With Bessel TPLSM, the fluorescence signal of a vessel becomes proportional to its size, which enables convenient intensity-based analysis of vessel dilation and constriction dynamics in large volumes. We observe entrainment of vasodilation and vasoconstriction with pupil diameter and measure 3D blood flow at 99 volumes/second. Demonstrating high-throughput monitoring of hemodynamics in the awake brain, we expect Bessel TPLSM to make broad impacts on neurovasculature research.


2021 ◽  
Vol 13 (5) ◽  
pp. 2950
Author(s):  
Su-Kyung Sung ◽  
Eun-Seok Lee ◽  
Byeong-Seok Shin

Climate change increases the frequency of localized heavy rains and typhoons. As a result, mountain disasters, such as landslides and earthworks, continue to occur, causing damage to roads and residential areas downstream. Moreover, large-scale civil engineering works, including dam construction, cause rapid changes in the terrain, which harm the stability of residential areas. Disasters, such as landslides and earthenware, occur extensively, and there are limitations in the field of investigation; thus, there are many studies being conducted to model terrain geometrically and to observe changes in terrain according to external factors. However, conventional topography methods are expressed in a way that can only be interpreted by people with specialized knowledge. Therefore, there is a lack of consideration for three-dimensional visualization that helps non-experts understand. We need a way to express changes in terrain in real time and to make it intuitive for non-experts to understand. In conventional height-based terrain modeling and simulation, there is a problem in which some of the sampled data are irregularly distorted and do not show the exact terrain shape. The proposed method utilizes a hierarchical vertex cohesion map to correct inaccurately modeled terrain caused by uniform height sampling, and to compensate for geometric errors using Hausdorff distances, while not considering only the elevation difference of the terrain. The mesh reconstruction, which triangulates the three-vertex placed at each location and makes it the smallest unit of 3D model data, can be done at high speed on graphics processing units (GPUs). Our experiments confirm that it is possible to express changes in terrain accurately and quickly compared with existing methods. These functions can improve the sustainability of residential spaces by predicting the damage caused by mountainous disasters or civil engineering works around the city and make it easy for non-experts to understand.


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