scholarly journals Wide field intravital imaging by two-photon-excitation digital-scanned light-sheet microscopy (2p-DSLM) with a high-pulse energy laser

2014 ◽  
Vol 5 (10) ◽  
pp. 3311 ◽  
Author(s):  
Atsushi Maruyama ◽  
Yusuke Oshima ◽  
Hiroko Kajiura-Kobayashi ◽  
Shigenori Nonaka ◽  
Takeshi Imamura ◽  
...  
Keyword(s):  
Pulse Energy ◽  
Light Sheet ◽  
Intravital Imaging ◽  
Wide Field ◽  
High Pulse Energy ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Photon Excitation ◽  
High Pulse ◽  
Energy Laser

scholarly journals Wide field light-sheet microscopy with lens-axicon controlled two-photon Bessel beam illumination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sota Takanezawa ◽  
Takashi Saitou ◽  
Takeshi Imamura
Keyword(s):  
High Speed ◽  
Bessel Beam ◽  
Light Sheet ◽  
Time Lapse ◽  
Whole Body ◽  
Wide Field ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Photon Excitation ◽  
Two Photon Excitation

AbstractTwo-photon excitation can lower phototoxicity and improve penetration depth, but its narrow excitation range restricts its applications in light-sheet microscopy. Here, we propose simple illumination optics, a lens-axicon triplet composed of an axicon and two convex lenses, to generate longer extent Bessel beams. This unit can stretch the beam full width at half maximum of 600–1000 μm with less than a 4-μm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and ~2–3-μm axial resolution is constructed and used to analyze the cellular dynamics over the whole body of medaka fish. We demonstrate long-term time-lapse observations over several days and high-speed recording with ~3 mm3 volume per 4 s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.

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 Improving Signal and Photobleaching Characteristics of Temporal Focusing Microscopy with the Increase in Pulse Repetition Rate

2019 ◽  
Vol 2 (3) ◽  
pp. 65
Author(s):  
Viktoras Lisicovas ◽  
Bala Murali Krishna Mariserla ◽  
Chakradhar Sahoo ◽  
Reuben T. Harding ◽  
Michael K. L. Man ◽  
...  
Keyword(s):  
Pulse Energy ◽  
Repetition Rate ◽  
Signal Intensity ◽  
High Repetition Rate ◽  
High Peak Power ◽  
High Pulse Energy ◽  
Two Photon ◽  
High Repetition ◽  
Laser Systems ◽  
High Pulse

Wide-field temporal focused (WF-TeFo) two-photon microscopy allows for the simultaneous imaging of a large planar area, with a potential order of magnitude enhancement in the speed of volumetric imaging. To date, low repetition rate laser sources with over half a millijoule per pulse have been required in order to provide the high peak power densities for effective two-photon excitation over the large area. However, this configuration suffers from reduced signal intensity due to the low repetition rate, saturation effects due to increased excitation fluences, as well as faster photobleaching of the fluorescence probe. In contrast, with the recent advent of high repetition rate, high pulse energy laser systems could potentially provide the advantages of high repetition rate systems that are seen in traditional two-photon microscopes, while minimizing the negatives of high fluences in WF-TeFo setups to date. Here, we use a 100 microjoule/high repetition rate (50–100 kHz) laser system to investigate the performance of a WF-TeFo two-photon microscope. While using micro-beads as a sample, we demonstrate a proportionate increase in signal intensity with repetition rate, at no added cost in photobleaching. By decreasing pulse intensity, via a corresponding increase in repetition rate to maintain fluorescence signal intensity, we find that the photobleaching rate is reduced by ~98.4%. We then image live C. elegans at a high repetition rate for 25 min. as a proof-of-principle. Lastly, we identify the steady state temperature increase as the limiting process in further increasing the repetition rate, and we estimate that repetition rate in the range between 0.5 and 5 MHz is ideal for live imaging with a simple theoretical model. With new generation low-cost fiber laser systems offering high pulse energy/high repetition rates in what is essentially a turn-key solution, we anticipate increased adoption of this microscopy technique by the neuroscience community.

scholarly journals A versatile, multi-laser twin-microscope system for light-sheet imaging

2019 ◽  
Author(s):  
Kevin Keomanee-Dizon ◽  
Scott E. Fraser ◽  
Thai V. Truong
Keyword(s):  
Near Infrared ◽  
Continuous Wave ◽  
Light Sheet ◽  
Biological Investigation ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Photon Excitation ◽  
Deep Imaging ◽  
Visible Lasers ◽  
Flexible Imaging

Light-sheet microscopy offers faster imaging and reduced phototoxicity in comparison to conventional point-scanning microscopy, making it a preferred technique for imaging biological dynamics for durations of hours or days. Such extended imaging sessions pose a challenge, as it reduces the number of specimens that can be imaged in a given day. Here we present an instrument, the flex-SPIM, that combines two independently controlled light-sheet microscope-twins, built so that they can share an ultrafast near-infrared laser and a bank of continuous-wave visible lasers, increasing throughput and decreasing cost. To permit a wide variety of specimens to be imaged, each microscope-twin provides flexible imaging parameters, including (i) operation in one-photon and/or two-photon excitation modes, (ii) delivery of one to three light-sheets via a trio of orthogonal excitation arms, (iii) sub-micron to micron imaging resolution, (iv) multicolor compatibility, and (v) upright and/or inverted detection geometry. We offer a detailed description of the flex-SPIM design to aid instrument builders who wish to construct and use similar systems. We demonstrate the instrument’s versatility for biological investigation by performing fast imaging of the beating heart in an intact zebrafish embryo, deep imaging of thick patient-derived tumor organoids, and gentle whole-brain imaging of neural activity in behaving larval zebrafish.

Enhancement of image quality in planar Airy light-sheet microscopy via subtraction method

2021 ◽  
Author(s):  
Suhui Deng ◽  
Liusong Yuan ◽  
Peiwei Cheng ◽  
Yuhao Wang ◽  
Mingping Liu
Keyword(s):  
Image Quality ◽  
Light Sheet ◽  
Large Field ◽  
Subtraction Method ◽  
Optical Sectioning ◽  
Two Photon ◽  
Light Sheet Microscopy ◽  
Airy Beam ◽  
Photon Excitation ◽  
Distribution Uniformity

Abstract The use of propagation-invariant Airy beams enables a light-sheet microscopy with a large field-of-view. Without relying upon two-photon excitation or deconvolution-based processing to eliminate out-of focus blur caused by the side lobes, here, we present how the subtraction method is applied to enhance the image quality in digital scanned light-sheet microscopy with Airy beam. In the proposed method, planar Airy beam with the symmetric transversal structure is used to excite the sample. A hollow Airy beam with zero intensity at the focal plane is created, which is mainly used to excite the out-of-focus signal. By scanning the sample twice with the normal planar Airy beam and the hollow Airy beam, digital post-processing of the obtained images by subtraction allows for the rejection of out-of-focus blur and improves the optical sectioning, the axial resolution and the intensity distribution uniformity of the light-sheet microscopy.

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