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Insight x3

Manufactured by Spectra-Physics
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Sourced in Germany, United States

The Insight X3 is a compact, high-performance femtosecond laser system designed for a variety of scientific and industrial applications. The Insight X3 produces ultrashort pulses with a pulse duration of less than 100 femtoseconds and a tunable wavelength range from 680 to 1300 nanometers. The laser system is designed for ease of use and reliability, making it a versatile tool for a wide range of research and industrial applications.

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Lab products found in correlation

Prairieview software, by Bruker (3 mentions) Ultima investigator, by Bruker (3 mentions) Cf175, by Nikon (2 mentions) Texas red dextran, by Thermo Fisher Scientific (2 mentions) Maitai deepsee ti sapphire laser, by Spectra-Physics (2 mentions) Fvmpe rs system, by Olympus (2 mentions) Fluoview software, by Olympus (2 mentions) Matlab, by MathWorks (2 mentions) 7 multiphoton microscope, by Zeiss (2 mentions) 2p ram, by Thorlabs (2 mentions) Bergamo 2, by Thorlabs (2 mentions) Lsm 880 microscope, by Zeiss (1 mentions) Ultima investigator, by Nikon (1 mentions) Fvmpe rs, by Olympus (1 mentions) Dextran, by Thermo Fisher Scientific (1 mentions) Ultima 4, by Bruker (1 mentions) 575dcxr, by Chroma Technology (1 mentions) 8khz resonant scanner, by Bruker (1 mentions) Model 7422p 40, by Hamamatsu Photonics (1 mentions) Fvmpe rs multiphoton laser scanning microscope, by Olympus (1 mentions)

30 protocols using insight x3

1

Two-Photon Imaging with Bessel Beam

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All imaging was performed under a home-built Bessel beam two-photon microscope with a 100  μm axial PSF. An axicon based Bessel module was used to generate an annular illumination focused at the back pupil plane of the objective (Olympus 25× , 1.0 NA). Microscope control and data acquisition were done using ScanImage46 (link) (Vidrio Technologies). Fluorescence was excited at 920 nm with a tunable Ti:Sapphire laser (Insight X3, Spectra Physics) with an electro-optic modulator (Conoptics 305-105-20) for fast power control. The excitation and emission paths were split by a primary dichroic (Semrock DI03-R785), and fluorescence was collected and detected by two cooled photomultiplier tubes (PMTs) (Hamamatsu H74422-40 and -50). The fluorescence was split by a secondary dichroic (Semrock FF555-DI03) and passed through either a red (Semrock FF01-607/70) or green (Semrock FF01-502/60) before being detected by the PMTs.
Giblin J., Kura S., Nunuez J.L., Zhang J., Kureli G., Jiang J., Boas D.A, & Chen I.A. (2023). High throughput detection of capillary stalling events with Bessel beam two-photon microscopy. Neurophotonics, 10(3), 035009.
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2

2-Photon Fluorescence Lifetime Imaging

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Fluorescence lifetime images were acquired using an upright laser-scanning fluorescence microscope (LSM880 microscope, Zeiss, Jena, Germany) with a 20× water immersion objective (numerical aperture 1.0, working distance 2.1 mm, XYZ Zeiss). Fluorescence was excited by 2-photon excitation using 120 fs light pulses with either λexc = 840 nm or 850 nm. Laser pulses were generated using a mode-locked Titan-Sapphire laser (InSight X3, Newport Spectra Physics, Darmstadt, Germany), with output powers ranging from 1.9 to 2.3 W for different excitation wavelengths, at a repetition rate of 80 MHz, while only a small fraction (typical: 10 mW) was applied to the sample.
Gonzalez M.M., Vizoso-Pinto M.G., Erra-Balsells R., Gensch T, & Cabrerizo F.M. (2024). In Vitro Effect of 9,9′-Norharmane Dimer against Herpes Simplex Viruses. International Journal of Molecular Sciences, 25(9), 4966.
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3

Two-Photon Imaging of Cortical Layers

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Images were acquired using a resonant scanning two-photon microscope (Ultima Investigator) at a 30 Hz frame rate and 512 × 512 pixel resolution through a 16× water-immersion lens (16×/0.8 numerical aperture; model CF175, Nikon). On separate days, either AC or PPC was imaged at a depth between 150 and 300 μm, corresponding to layers 2/3 of cortex. For AC imaging, the objective was rotated 35–45° from vertical, and for PPC imaging, it was rotated to 5–15° from vertical, matching the angle of the cranial window implant. Fields of view were 500 μm2 and contained 187 ± 95 neurons, 20 ± 10 (mean ± SD) of which were classified as SOM neurons. Excitation light was provided by a femtosecond infrared (IR) laser (Insight X3, Spectra-Physics) tuned to 920 nm. Green and red wavelengths were separated through a 565 nm low-pass filter before passing through bandpass filters (catalog #ET525/70 and #ET595/50, Chroma). PrairieView software (version 5.5; Bruker) was used to control the microscope.
Khoury C.F., Fala N.G, & Runyan C.A. (2023). Arousal and Locomotion Differently Modulate Activity of Somatostatin Neurons across Cortex. eNeuro, 10(5), ENEURO.0136-23.2023.
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4

Intravital Imaging of Mouse Calvarial Bone Marrow

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Mice were anesthetized and placed on a custom stereotactic frame. During imaging, anesthesia was maintained with ∼1.5% isoflurane in 100% oxygen, with small adjustments to maintain the respiratory rate at ∼1 Hz. To fluorescently label the microvasculature, Texas red dextran (40 μl, 2.5%, molecular weight (MW) = 70,000 kDA, Thermo Fisher Scientific) in saline was injected retro-orbitally immediately before imaging. Three-dimensional data sets of the calvarial bone marrow, meninges, meningeal vasculature and CSF transport were obtained using a custom-built two-photon excitation microscope. Imaging was done using 830 nm, 120 fs pulses from a Ti:Sapphire laser oscillator (Spectra-Physics InSight X3). The laser beam was scanned by polygon scanners (30 frames s–1) and focused into the sample using a 60x water-immersion objective lens for high-resolution imaging (numerical aperture of 1.1, Olympus). The emitted fluorescence was detected on photomultiplier tubes through the following emission filters: 400/60 nm for second harmonic generation (SHG), 525/50 nm for Alexa488/FITC and 605/50 nm for Texas red. Laser scanning and data acquisition were controlled by custom-built software. Stacks of images were spaced at 1 μm axially.
Pulous F.E., Cruz-Hernández J.C., Yang C., Kaya Ζ., Paccalet A., Wojtkiewicz G., Capen D., Brown D., Wu J.W., Schloss M.J., Vinegoni C., Richter D., Yamazoe M., Hulsmans M., Momin N., Grune J., Rohde D., McAlpine C.S., Panizzi P., Weissleder R., Kim D.E., Swirski F.K., Lin C.P., Moskowitz M.A, & Nahrendorf M. (2022). Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis. Nature neuroscience, 25(5), 567-576.
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5

Intravital Imaging of Mouse Calvarial Bone Marrow

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Mice were anesthetized and placed on a custom stereotactic frame. During imaging, anesthesia was maintained with ∼1.5% isoflurane in 100% oxygen, with small adjustments to maintain the respiratory rate at ∼1 Hz. To fluorescently label the microvasculature, Texas red dextran (40 μl, 2.5%, molecular weight (MW) = 70,000 kDA, Thermo Fisher Scientific) in saline was injected retro-orbitally immediately before imaging. Three-dimensional data sets of the calvarial bone marrow, meninges, meningeal vasculature and CSF transport were obtained using a custom-built two-photon excitation microscope. Imaging was done using 830 nm, 120 fs pulses from a Ti:Sapphire laser oscillator (Spectra-Physics InSight X3). The laser beam was scanned by polygon scanners (30 frames s–1) and focused into the sample using a 60x water-immersion objective lens for high-resolution imaging (numerical aperture of 1.1, Olympus). The emitted fluorescence was detected on photomultiplier tubes through the following emission filters: 400/60 nm for second harmonic generation (SHG), 525/50 nm for Alexa488/FITC and 605/50 nm for Texas red. Laser scanning and data acquisition were controlled by custom-built software. Stacks of images were spaced at 1 μm axially.
Pulous F.E., Cruz-Hernández J.C., Yang C., Kaya Ζ., Paccalet A., Wojtkiewicz G., Capen D., Brown D., Wu J.W., Schloss M.J., Vinegoni C., Richter D., Yamazoe M., Hulsmans M., Momin N., Grune J., Rohde D., McAlpine C.S., Panizzi P., Weissleder R., Kim D.E., Swirski F.K., Lin C.P., Moskowitz M.A, & Nahrendorf M. (2022). Cerebrospinal fluid can exit into the skull bone marrow and instruct cranial hematopoiesis in mice with bacterial meningitis. Nature neuroscience, 25(5), 567-576.
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6

Two-Photon Microscopy Imaging Protocol

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All images were collected by an Olympus FVMPE-RS system (Olympus, Center Valley, PA) using an Olympus 25× water objective (XLPLN25XWMP2, 1.05NA). The system was equipped with two two-photon lasers: Spectra-Physics InSightX3 (680nm-1300nm, Spectra-Physics, Santa Clara, CA) and Spectra-Physics MaiTai DeepSee Ti:Sapphire laser (690nm-1040nm). There were four Photon Multiplier Tubes (PMTs) and two filter cubes (Blue/Green cube: 420-460nm/495-540nm, Red/Far Red cube: 575-630nm/645-685nm) for multi-color imaging. A galvanometer scanner was used for scanning, and all images were acquired at ~1 frame/s. PMT gains for all imaging were used between 500 and 700 airy units in the Olympus Fluoview software.
Emo K., Reilly E.C., Sportiello M., Yang H, & Topham D.J. (2020). T cell and chemokine receptors differentially control CD8 T cell motility behavior in the infected airways immediately before and after virus clearance in a primary infection. PLoS ONE, 15(8), e0227157.
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7

Multimodal 2P Imaging of Microvascular Flow

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An Olympus FVMPE-RS system equipped with two 2-photon lasers: Spectra-Physics InSightX3 (680nm-1300nm) and Spectra-Physics MaiTai DeepSee Ti:Sapphire laser (690nm-1040nm), and a 25X water objective (XLPLN25XWMP2, 1.05NA), was used for high resolution imaging. With the laser tuned to 780nm, images were acquired from resonant scanners at a resolution of 512x512 pixels with the z-step size of 5 μm. The fluorescence of GFP, RFP, far-red RFP and Second Harmonic Generation (SHG) signals were collected with a 517/23-nm, a 605/25-nm, a 665/20nm, and a 390/20-nm bandpass filters (Semrock), respectively. The 2D slice viewing and 3D reconstruction of the defect were performed in Imaris (Bitplane Inc., Concord, MA) and Amira (Visage Imaging, Berlin, Germany) image analysis software. Red blood cell (RBC) velocity analyses were performed using a water-immersion objective (×25, NA 1.05) for line-scan measurements. These measurements utilized 640x640 pixel images with a pixel dwell of 10us/pixel. RBC velocity was calculated based on Radon transformation and an automated image-processing algorithm provided by MATLAB [26 ]. Vessel diameters were measured manually using ImageJ (National Institutes of Health).
Zhai Y., Schilling K., Wang T., Khatib M.E., Vinogradov S., Brown E, & Zhang X. (2021). Spatiotemporal Blood Vessel Specification at the Osteogenesis and Angiogenesis Interface of Biomimetic Nanofiber-Enabled Bone Tissue Engineering. Biomaterials, 276, 121041.
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8

Hyperspectral SRS Imaging Protocol

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Hyperspectral SRS imaging is performed under a commercial system constructed by sending a dual-output femtosecond laser system (InSight X3, Spectra-Physics) through an integrated Spectral Focusing Timing and Recombination Unit (SF-TRU, Newport Corporation) (38 ) and coupled into a multiphoton laser scanning microscope (FVMPE-RS, Olympus). The instrumentation and imaging condition are described in detail in SI Appendix.
Qian N., Gao X., Lang X., Deng H., Bratu T.M., Chen Q., Stapleton P., Yan B, & Min W. (2024). Rapid single-particle chemical imaging of nanoplastics by SRS microscopy. Proceedings of the National Academy of Sciences of the United States of America, 121(3), e2300582121.
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9

Femtosecond Laser-Driven ST Light Sheet

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A femtosecond laser (InSight X3, Spectra-Physics) is used to generate the ST light sheet with 150 fs pulses at a repetition rate of 80 MHz. The excitation wavelength for this work is centered at 800 nm, and the laser is spectrally adjustable between 680 and 1300 nm. For the light sheet used in the paper, we use the subluminal regime with θ = 44.98° corresponds to spectral tilt angles in the range of 0° < θ < 45°. The superluminal of ST light sheet corresponds to spectral tilt angles of 45° < θ < 180°, with a positive group velocity (vg). We measured the bandwidth of the ST light sheet ∆λ = 2 nm (see Fig. S1 in the supplemental material). These variables are essential for keeping the ST light sheet and avoiding spatial spreading.
Diouf M., Lin Z., Harling M, & Toussaint, KC J.r. (2022). Demonstration of speckle resistance using space–time light sheets. Scientific Reports, 12, 14064.
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10

Multimodal Microscopy Imaging Protocol

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All images were collected by an Olympus FVMPE-RS system (Olympus, Center Valley, PA) using Olympus 25´ water objective (XLPLN25XWMP2, 1.05NA). The system was equipped with two two-photon lasers: Spectra-Physics InSightX3 (680nm-1300nm, Spectra-Physics, Santa Clara, CA) and Spectra-Physics MaiTai DeepSee Ti:Sapphire laser (690nm-1040nm). There were four Photon Multiplier Tubes (PMTs) and two filter cubes (Blue/Green cube: 420-460nm/495-540nm, red/fRed cube: 575-630nm/645-685nm) for multi-color imaging.
Galvonometer scanner was used for scanning, and all images were acquired at ~1frame/s. PMT gains for all imaging were used between 550 and 700 a.u. in the Olympus Fluoview software. All images were registered within Matlab (MathWorks) using the registration code provided by the Center for Integrated Research Computing (https://github.com/TophamLab/Registration) and were analyzed and visualized using Imaris (Bitplane) software.
Reilly E.C., Lambert-Emo K., Buckley P.M., Reilly N., Chaves F.A., Yang H., Oakes P.W., & Topham D.J. (2020). TRMIntegrins CD103 and CD49a Differentially Support Adherence and Motility After Resolution of Influenza Virus Infection.
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