scholarly journals Optimization of the excitation light sheet in selective plane illumination microscopy

2015 ◽  
Vol 6 (3) ◽  
pp. 881 ◽  
Author(s):  
Liang Gao
Keyword(s):  
Light Sheet ◽  
Excitation Light ◽  
Selective Plane Illumination Microscopy

scholarly journals A method to keep the excitation light sheet in focus in selective plane illumination microscopy

2017 ◽  
Author(s):  
Liang Gao
Keyword(s):  
3D Imaging ◽  
Light Sheet ◽  
Axial Position ◽  
Excitation Light ◽  
Selective Plane Illumination Microscopy

AbstractKeeping the excitation light sheet in focus is critical in selective plane illumination microscopy (SPIM) to ensure its 3D imaging ability. Unfortunately, an effective method that can be used in SPIM on general biological specimens to find the axial position of the excitation light sheet and keep it in focus is barely available. Here, we present a method to solve the problem. We investigate its mechanism and demonstrate its performance on a lattice light sheet microscope.

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

scholarly journals Tiling light sheet selective plane illumination microscopy using discontinuous light sheets

2018 ◽  
Author(s):  
Liang Gao
Keyword(s):  
Spatial Resolution ◽  
3D Imaging ◽  
High Spatial Resolution ◽  
Image Data ◽  
Image Plane ◽  
Light Sheet ◽  
Selective Plane Illumination Microscopy ◽  
Novel Method ◽  
Technical Details ◽  
Imaging Speed

AbstractTiling light sheet selective plane illumination microscopy (TLS-SPIM) improves 3D imaging ability of SPIM by using a real-time optimized tiling light sheet. However, the imaging speed decreases, and size of the raw image data increases proportionally to the number of tiling positions in TLS-SPIM. The decreased imaging speed and the increased raw data size could cause significant problems when TLS-SPIM is used to image large specimens at high spatial resolution. Here, we present a novel method to solve the problem. Discontinuous light sheets created by scanning coaxial beam arrays synchronized with camera exposures are used for 3D imaging to decrease the number of tiling positions required at each image plane without sacrificing the spatial resolution. We investigate the performance of the method via numerical simulation and discuss the technical details of the method.

scholarly journals Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy

2020 ◽  
Vol 11 (8) ◽  
pp. 4651
Author(s):  
Giuseppe de Vito ◽  
Pietro Ricci ◽  
Lapo Turrini ◽  
Vladislav Gavryusev ◽  
Caroline Müllenbroich ◽  
...  
Keyword(s):  
Fluorescence Emission ◽  
Light Sheet ◽  
Light Polarization ◽  
Excitation Light ◽  
Two Photon ◽  
Light Sheet Microscopy

scholarly journals Tiling light sheet selective plane illumination microscopy using discontinuous light sheets

2019 ◽  
Vol 27 (23) ◽  
pp. 34472 ◽  
Author(s):  
Dongyue Wang ◽  
Yuxiao Jin ◽  
Ruili Feng ◽  
Yanlu Chen ◽  
Liang Gao
Keyword(s):  
Light Sheet ◽  
Selective Plane Illumination Microscopy

scholarly journals μSPIM: A Software Platform for Selective Plane Illumination Microscopy

2020 ◽  
Author(s):  
Daniel Saska ◽  
Paul Pichler ◽  
Chen Qian ◽  
Chrysia Pegasiou ◽  
Christopher L. Buckley ◽  
...  
Keyword(s):  
Optical Design ◽  
Light Sheet ◽  
Volumetric Imaging ◽  
Larval Zebrafish ◽  
Selective Plane Illumination Microscopy ◽  
Spatio Temporal ◽  
Set Up ◽  
Control And Synchronization ◽  
The Brain ◽  
Camera Shutter

AbstractSelective Plane Illumination Microscopy (SPIM) is a fluorescence imaging technique that allows volumetric imaging at high spatio-temporal resolution to monitor neural activity in live organisms such as larval zebrafish. A major challenge in the construction of a custom SPIM microscope is the control and synchronization of the various hardware components. Here we present a control toolset, μSPIM, built around the open-source MicroManager platform that has already been widely adopted for the control of microscopy hardware. Installation of μSPIM is relatively straightforward, involving a single C++ executable and a Java-based extension to Micro-Manager. Imaging protocols are defined through the μSPIM extension to Micro-Manager. The extension then synchronizes the camera shutter with the galvanometer mirrors to create a light-sheet that is scanned in the z-dimension, in synchrony with the imaging objective, to produce volumetric recordings. A key advantage of μSPIM is that a series of calibration procedures optimizes acquisition for a given set-up making it relatively independent of the optical design of the microscope, or the hardware used to build it. Two laser illumination arms can be used while also allowing for the introduction of illumination masks. μSPIM allows imaging of calcium activity throughout the brain of larval zebrafish at rates of 100 planes per second with single cell resolution as well as slower imaging to reconstruct cell populations, for example, in the cleared brains of mice.

scholarly journals Fast, multicolor 3-D imaging of brain organoids with a new single-objective two-photon virtual light-sheet microscope

2018 ◽  
Author(s):  
Irina Rakotoson ◽  
Brigitte Delhomme ◽  
Philippe Djian ◽  
Andreas Deeg ◽  
Maia Brunstein ◽  
...  
Keyword(s):  
Spatial Resolution ◽  
Three Dimensional ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Confocal Microscope ◽  
Large Field ◽  
Excitation Light ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Functional Features

ABSTRACTHuman inducible pluripotent stem cells (hiPSCs) hold a large potential for disease modeling. hiPSC-derived human astrocyte and neuronal cultures permit investigations of neural signaling pathways with subcellular resolution. Combinatorial cultures, and three-dimensional (3-D) embryonic bodies enlarge the scope of investigations to multi-cellular phenomena. A the highest level of complexity, brain organoids that – in many aspects – recapitulate anatomical and functional features of the developing brain permit the study of developmental and morphological aspects of human disease. An ideal microscope for 3-D tissue imaging at these different scales would combine features from both confocal laser-scanning and light-sheet microscopes: a micrometric optical sectioning capacity and sub-micrometric spatial resolution, a large field of view and high frame rate, and a low degree of invasiveness, i.e., ideally, a better photon efficiency than that of a confocal microscope. In the present work, we describe such an instrument that belongs to the class of two-photon (2P) light-sheet microsocpes. Its particularity is that – unlike existing two- or three-lens designs – it is using a single, low-magnification, high-numerical aperture objective for the generation and scanning of a virtual light sheet. The microscope builds on a modified Nipkow-Petran spinning-disk scheme for achieving wide-field excitation. However, unlike the common Yokogawa design that uses a tandem disk, our concept combines micro lenses, dichroic mirrors and detection pinholes on a single disk. This design, advantageous for 2P excitation circumvents problems arising with the tandem disk from the large wavelength-difference between the infrared excitation light and visible fluorescence. 2P fluorescence excited in by the light sheet is collected by the same objective and imaged onto a fast sCMOS camera. We demonstrate three-dimensional imaging of TO-PRO3-stained embryonic bodies and of brain organoids, under control conditions and after rapid (partial) transparisation with triethanolamine and /ormamide (RTF) and compare the performance of our instrument to that of a confocal microscope having a similar numerical aperture. 2P-virtual light-sheet microscopy permits one order of magnitude faster imaging, affords less photobleaching and permits better depth penetration than a confocal microscope with similar spatial resolution.

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