A versatile, multi-laser twin-microscope system for light-sheet 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.

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

Biophysical Journal ◽

10.1016/j.bpj.2011.11.1065 ◽

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

Biomedical Optics Express ◽

10.1364/boe.5.003311 ◽

2014 ◽

Vol 5 (10) ◽

pp. 3311 ◽

Cited By ~ 11

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

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

Nature Communications ◽

10.1038/s41467-021-23249-y ◽

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.

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

Journal of Optics ◽

10.1088/2040-8986/ac4437 ◽

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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Two-Photon near Infrared Excitation in Living Cells

Journal of Near Infrared Spectroscopy ◽

10.1255/jnirs.97 ◽

1997 ◽

Vol 5 (1) ◽

pp. 27-34 ◽

Cited By ~ 4

Author(s):

Karsten König

Keyword(s):

Near Infrared ◽

Continuous Wave ◽

Cell Damage ◽

Local Heating ◽

Average Power ◽

Two Photon ◽

Photon Excitation ◽

Non Linear ◽

Linear Effects ◽

Two Photon Excitation

Non-linear effects due to two-photon near infrared (NIR) excitation of endogenous and exogenous cellular chromophores allow novel techniques in tissue, cell and biomolecule diagnostics, as well as in intracellular micromanipulation (e.g. intracellular photochemistry). Two-photon NIR excitation may also result in cell damage effects. The high photon intensities (1024 photons cm−2 s−1) required for non-resonant two-photon excitation can be achieved by diffraction-limited focusing of continuous wave (cw) laser beams (cw microbeams) with powers in the mW range. For example, NIR traps (“laser tweezers”) used as force transducers and micromanipulation tools in cellular and molecular biology are sources of two-photon excitation. NIR traps can induce two-photon excited visible fluorescence and, in the case of <800 nm-traps, UVA-like cell damage. Multimode cw microbeams may enhance non-linear effects due to longitudinal mode-beating. To perform high scan rate two-photon fluorescence imaging, the application of ultrashort laser pulses of moderate peak power but low average power (pulsed microbeams) is required. In NIR femtosecond microscopes, non-destructive imaging of two-photon excited fluorophores in various human and culture cells was demonstrated for <2 mW average powers, <200 mW peak powers and 400 GW cm−2 intensities (700–800 nm, ∼150 fs, ∼100 MHz). However, higher average power levels may result in failed cell reproduction and cell death due to intracellular optical breakdown. In addition, destructive transient local heating and μN force generation may occur.

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Two-Photon–NIR-II Antimicrobial Graphene-Nanoagent for Ultraviolet–NIR Imaging and Photoinactivation

10.21203/rs.3.rs-519688/v1 ◽

2021 ◽

Author(s):

WEN-SHUO KUO ◽

Chia-Yuan Chang ◽

Ping-Ching Wu ◽

Jiu-Yao Wang

Keyword(s):

Photodynamic Therapy ◽

Quantum Yield ◽

Chemical Modification ◽

Near Infrared ◽

Photon Absorption ◽

Excitation Wavelength ◽

Strong Electron ◽

Two Photon ◽

Photon Excitation ◽

Two Photon Excitation

Abstract BackgroundNitrogen doping and amino-group functionalization, which result in strong electron donation, can be achieved through chemical modification. Large π-conjugated systems of graphene quantum dot (GQD)-based materials acting as electron donors can be chemically manipulated with low two-photon excitation energy in a short photoexcitation time for improving the charge transfer efficiency of sorted nitrogen-doped amino acid–functionalized GQDs (sorted amino-N-GQDs). ResultsIn this study, a self-developed femtosecond Ti-sapphire laser optical system (222.7 nJ pixel−1 with 100-170 scans, approximately 0.65-1.11 s of total effective exposure times; excitation wavelength: 960 nm in the near-infrared II region) was used for chemical modification. The sorted amino-N-GQDs exhibited enhanced two-photon absorption, post-two-photon excitation stability, two-photon excitation cross-section, and two-photon luminescence through the radiative pathway. The lifetime and quantum yield of the sorted amino-N-GQDs decreased and increased, respectively. Furthermore, the sorted amino-N-GQDs exhibited excitation-wavelength-independent photoluminescence in the near-infrared region and generated reactive oxygen species after two-photon excitation. An increase in the size of the sorted amino-N-GQDs boosted photochemical and electrochemical efficacy and resulted in high photoluminescence quantum yield and highly efficient two-photon photodynamic therapy. ConclusionThe sorted dots can be used in two-photon contrast probes for tracking and localizing analytes during two-photon imaging in a biological environment and for conducting two-photon photodynamic therapy for eliminating infectious microbes.

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