scholarly journals Multiview Deconvolution Two-photon Laser Scanning Microscopy

2020 ◽  
Author(s):  
Dimitrios Kapsokalyvas ◽  
Rodrigo Rosas ◽  
Rob Janssen ◽  
Jo Vanoevelen ◽  
Martin Strauch ◽  
...  
Keyword(s):  
Second Harmonic ◽  
Light Sheet ◽  
Laser Scanning Microscopy ◽  
Three Dimensions ◽  
Scanning Microscopy ◽  
Fluorescence Signal ◽  
Microscopy Imaging ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
3D Image Reconstruction

Abstract Imaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, two-photon and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy - imaging the sample from different angles followed by 3D image reconstruction - was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with two-photon microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least 3-fold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

scholarly journals Multiview deconvolution approximation multiphoton microscopy of tissues and zebrafish larvae

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Dimitrios Kapsokalyvas ◽  
Rodrigo Rosas ◽  
Rob W. A. Janssen ◽  
Jo M. Vanoevelen ◽  
Miranda Nabben ◽  
...  
Keyword(s):  
Second Harmonic ◽  
Multiphoton Microscopy ◽  
Light Sheet ◽  
Three Dimensions ◽  
Fluorescence Signal ◽  
Label Free ◽  
Microscopy Imaging ◽  
Isotropic Resolution ◽  
Light Sheet Microscopy ◽  
3D Image Reconstruction

AbstractImaging in three dimensions is necessary for thick tissues and small organisms. This is possible with tomographic optical microscopy techniques such as confocal, multiphoton and light sheet microscopy. All these techniques suffer from anisotropic resolution and limited penetration depth. In the past, Multiview microscopy—imaging the sample from different angles followed by 3D image reconstruction—was developed to address this issue for light sheet microscopy based on fluorescence signal. In this study we applied this methodology to accomplish Multiview imaging with multiphoton microscopy based on fluorescence and additionally second harmonic signal from myosin and collagen. It was shown that isotropic resolution was achieved, the entirety of the sample was visualized, and interference artifacts were suppressed allowing clear visualization of collagen fibrils and myofibrils. This method can be applied to any scanning microscopy technique without microscope modifications. It can be used for imaging tissue and whole mount small organisms such as heart tissue, and zebrafish larva in 3D, label-free or stained, with at least threefold axial resolution improvement which can be significant for the accurate quantification of small 3D structures.

Second Harmonic Imaging Microscopy: A New Non-Linear Optical Modality for Cell Membrane Physiology

2000 ◽  
Vol 6 (S2) ◽  
pp. 810-811
Author(s):  
Paul Campagnola ◽  
Aaron Lewis ◽  
Leslie M. Loew
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Second Harmonic ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Photon Absorption ◽  
Photon Flux ◽  
Two Photon ◽  
Non Linear ◽  
Linear Optical

Confocal microscopy is an excellent high resolution method to image fluorescently labeled cells. However, the use of confocal microscopy to monitor physiological events, such as membrane potential changes, in living cells is hampered by photobleaching and phototoxicity. To reduce the collateral damage from excitation of fluorescent probes outside the optical slice, Webb and co-workers introduced the use of two-photon excited (TPE) fluorescence in laser scanning microscopy.1 Two-photon absorption depends on the square of the incident light intensity; this has the effect of confining excitation to the plane of focus where the photon flux density is greatest. The wavelength of the exciting light is in the near infrared facilitating penetration of thick tissues. Due to these significant advantages this methodology is rapidly gaining popularity as a tool for live cell and tissue imaging.To further exploit non-linear optical processes in laser scanning microscopy, we have developed surface second harmonic generation (SHG) as a powerful new imaging modality.

scholarly journals High-speed volumetric two-photon fluorescence imaging of neurovascular dynamics

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiang Lan Fan ◽  
Jose A. Rivera ◽  
Wei Sun ◽  
John Peterson ◽  
Henry Haeberle ◽  
...  
Keyword(s):  
Blood Flow ◽  
High Speed ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Fluorescence Signal ◽  
Imaging Method ◽  
Spatiotemporal Resolution ◽  
Two Photon

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.

scholarly journals Advanced Label-Free Laser Scanning Microscopy and Its Biological Imaging Application

2021 ◽  
Vol 11 (3) ◽  
pp. 1002
Author(s):  
Xue Wang ◽  
Xinchao Lu ◽  
Chengjun Huang
Keyword(s):  
Laser Scanning ◽  
Second Harmonic ◽  
Biological Tissues ◽  
Biological Imaging ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Localized Surface Plasmon ◽  
Label Free ◽  
Coherent Raman ◽  
Surface Plasmon Microscopy

By eliminating the photodamage and photobleaching induced by high intensity laser and fluorescent molecular, the label-free laser scanning microscopy shows powerful capability for imaging and dynamic tracing to biological tissues and cells. In this review, three types of label-free laser scanning microscopies: laser scanning coherent Raman scattering microscopy, second harmonic generation microscopy and scanning localized surface plasmon microscopy are discussed with their fundamentals, features and recent progress. The applications of label-free biological imaging of these laser scanning microscopies are also introduced. Finally, the performance of the microscopies is compared and the limitation and perspectives are summarized.

scholarly journals Two-photon laser scanning microscopy with electrowetting-based prism scanning

2017 ◽  
Vol 8 (12) ◽  
pp. 5412 ◽  
Author(s):  
Omkar D. Supekar ◽  
Baris N. Ozbay ◽  
Mo Zohrabi ◽  
Philip D. Nystrom ◽  
Gregory L. Futia ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Scanning Microscopy ◽  
Two Photon

scholarly journals Characterization of Scattering Effects in Phantom Samples using Single and Two-Photon Excitation Light Sheet Microscopy

2012 ◽  
Vol 102 (3) ◽  
pp. 195a-196a
Author(s):  
Zeno Lavagnino ◽  
Francesca Cella Zanacchi ◽  
Emiliano Ronzitti ◽  
Ivan Coto Hernandez ◽  
Alberto Diaspro
Keyword(s):  
Light Sheet ◽  
Excitation Light ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Photon Excitation ◽  
Scattering Effects ◽  
Two Photon Excitation
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