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 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 Efficiency of Plasmon-Induced Dual-Mode Fluorescence Enhancement upon Two-Photon Excitation

Nanomaterials ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3334
Author(s):  
Maria A. Shokova ◽  
Vladimir E. Bochenkov
Keyword(s):  
Mode Coupling ◽  
Rational Design ◽  
Fluorescence Enhancement ◽  
Whole Body ◽  
Whole Body Imaging ◽  
Dual Mode ◽  
Excitation Rate ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

Anisotropic noble metal nanoparticles supporting more than one localized surface plasmon resonance can be tailored for efficient dual-mode fluorescence enhancement by ensuring an adequate coupling to both absorption and emission bands of fluorophores. This approach is naturally extended to two-photon excitation fluorescence, where a molecule is excited by simultaneous nonlinear absorption of two photons. However, the relative impact of plasmon coupling to excitation and emission on the overall fluorescence enhancement can be very different in this case. Here, by using the finite-difference time-domain method, we study the two-photon excitation fluorescence of near-infrared fluorescent protein (NirFP) eqFP670, which is the most red-shifted NirFP to date, in proximity to a silver nanobar. By optimizing the length and aspect ratio of the particle, we reach a fluorescence enhancement factor of 103. We show that the single mode coupling regime with highly tuned near-field significantly outperforms the dual-mode coupling enhancement. The plasmon-induced amplification of the fluorophore’s excitation rate becomes of utmost importance due to its quadratic dependence on light intensity, defining the fluorescence enhancement upon two-photon excitation. Our results can be used for the rational design of hybrid nanosystems based on NirFP and plasmonic nanoparticles with greatly improved brightness important for developing whole-body imaging techniques.

High Speed Two Photon Excitation Microscopy in Live Cell Imaging using Image Correlation Spectroscopy (ICS)

2001 ◽  
Vol 7 (S2) ◽  
pp. 22-23
Author(s):  
P. W. Wiseman ◽  
J. C. Bouwer ◽  
S. Peltier ◽  
M. H. Ellisman
Keyword(s):  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Live Cell ◽  
Image Correlation ◽  
Scanning System ◽  
Optical Sectioning ◽  
Two Photon ◽  
Photon Excitation ◽  
Two Photon Excitation

For live-cell imaging, two-photon excitation microscopy (TPEM) is proving to be a significant technological advancement. The unique features offered by TPEM are the ability to image thick sections, excellent optical sectioning capabilities, low damage to living cells, and less out of focus fluorescence and out of focus photobleaching. of these features, the most useful for the biological microscopist, is optical sectioning. Optical sectioning is an intrinsic property of the two-photon process, whereby, two infrared (IR) photons are absorbed quickly to excite a single UV/blue transition. The probability for exciting a two photon transition is proportional to the instantaneous excitation intensity squared. Therefore, for a focused laser beam, only light at the focal point of the excitation beam excites a fluorescent transition. Thus, the need for confocal apertures and time consuming deconvolution algorithms are, for the most part, eliminated.We have continued to develop and enhance our ability to perform high-speed, two-photon excitation fluorescence microscopy. in 1998, we successfully deployed a prototype, video-rate twophoton laser scanning system (30 frames/sec or faster at reduced scan width) developed with support from Nikon Corporation. That system was built upon a Nikon RCM 8000 confocal microscope.

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.

scholarly journals 3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation

2012 ◽  
Vol 20 (24) ◽  
pp. 26219 ◽  
Author(s):  
Heejin Choi ◽  
Dimitrios S. Tzeranis ◽  
Jae Won Cha ◽  
Philippe Clémenceau ◽  
Sander J. G. de Jong ◽  
...  
Keyword(s):  
Wide Field ◽  
Phosphorescence Lifetime ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Photon Excitation ◽  
Temporal Focusing ◽  
Phosphorescence Lifetime Imaging ◽  
Fluorescence And Phosphorescence ◽  
Two Photon Excitation
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