One day following BM biopsy, mice were anesthetized using KXA (Ketamine, Xyalzine, Acepromazine) solution (4 μl/g) and secured on an externally heated imaging plate in a supine position. The medial region of tibia (contralateral to the femur used for biopsy) was surgically exposed and thinned to approximately 200 μm thickness using a mechanical microdrill. The leg was immobilized using a custom-built apparatus which contained an opening for the drilled bone to be exposed to the microscope objective. Images were collected on an Olympus FV1000-MPE upright laser scanning microscope fitted with a 25× 1.05NA water immersion objective and Mai Tai DeepSee Ti:Sapphire laser (Spectra-Physics) for two-photon excitation. For photoactivation experiments, selected cells were exposed to a brief pulse of laser tuned to 830 nm then subsequently imaged at 920 – 940 nm. Animal were kept under anesthesia using isoflurane inhalation during the entire intravital imaging session. 100–200 μl of lactated Ringer’s solution was periodically administered by subcutaneous injection to ensure proper hydration.
Maitai deepsee ti sapphire laser
Manufactured by Spectra-Physics
Sourced in Canada
The MaiTai DeepSee Ti:Sapphire laser is a tunable, mode-locked laser that produces ultrashort pulses of light in the near-infrared wavelength range. It is designed for use in various applications, including multiphoton microscopy, nonlinear spectroscopy, and other research areas requiring a high-performance, reliable laser source.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview software, by Olympus (2 mentions)
Fvmpe rs system, by Olympus (2 mentions)
Insight x3, by Spectra-Physics (2 mentions)
Fv1000 mpe upright laser scanning microscope, by Spectra-Physics (1 mentions)
Axioimager z1 platform, by Zeiss (1 mentions)
Af6000, by Leica (1 mentions)
Dfc500 camera, by Leica (1 mentions)
Las software, by Leica (1 mentions)
M205fa combi scope, by Leica (1 mentions)
Orca er, by Hamamatsu Photonics (1 mentions)
Zen 2012, by Zeiss (1 mentions)
Plan apochromat 10x 0.45 m27 objective, by Zeiss (1 mentions)
Fv1000 mpe microscope, by Olympus (1 mentions)
Lsm 710, by Zeiss (1 mentions)
Matlab, by MathWorks (1 mentions)
8 protocols using maitai deepsee ti sapphire laser
1
Two-Photon Intravital Imaging of Bone Microenvironment
2
Customized Multipath Optical Setup for Imaging and Stimulation
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Our customized optical setup was based on a AxioImager Z1 platform (Zeiss, Thornwood, NY) fitted with an Ultima dual path scan head containing two pairs of galvanometric mirrors (Bruker, Middleton, WI) for separate control of imaging and stimulating optical pathways (Figure 1—figure supplement 1 ). The imaging pathway used a Chameleon Ti:sapphire laser tuned to 920 nm (Coherent, Santa Clara, CA). The stimulation pathway employed a MaiTai DeepSee Ti:sapphire laser tuned to 880 nm (SpectraPhysics, Santa Clara, CA). The spectral broadening necessary for temporal focusing was achieved on this path by placing a 300 line/mm diffraction grating (Thorlabs, Newton, NJ) approximately 1 m away from the galvanometric mirrors; the laser spot on the grating was imaged onto the plane between the mirrors using a 500 mm focal length lens (Thorlabs) placed 500 mm away from the grating along the path of the first diffraction order from the grating. The stimulation laser was recombined with the imaging laser in the scan head using a 900 nm long-pass dichroic mirror (Chroma, Bellows Falls, VT). The power applied by either laser was controlled with a separate Pockels cell (Conoptics, Danbury, CT).
3
Confocal Imaging of Embryonic Development
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Embryos were analyzed under a M205FA combi-scope (Leica Microsystems CMS GmbH, Wetzlar, Germany). Representative images of at least four embryos in each group per experiment were obtained using brightfield DFC290 (Leica Microsystems CMS GmbH) and/or fluorescent ORCA-ER (Hamamatsu, Hamamatsu City, Japan) cameras. Sections were imaged under a DM6000B upright microscope (Leica Microsystems CMS GmbH) equipped with a DFC500 camera (Leica Microsystems CMS GmbH). The images were processed and analyzed using Adobe Photoshop (San Jose, CA, USA), LAS software (Leica Microsystems CMS GmbH), and/or AF6000 software (Leica Microsystems CMS GmbH).
Time-lapse imaging of Tg(foxd3:GFP) embryos was performed as previously described.23 (link) Briefly, the embryos were imaged using a TCS SP5 MP multiphoton microscope (Leica Microsystems CMS GmbH) fitted with a Mai Tai DeepSee Ti-Sapphire laser (Spectra-Physics; Newport Corp., Irvine, CA, USA). The images were obtained every 20 minutes during the described time frame. Each time-lapse experiment was repeated three or four times. Images and movies included are representative of all experiments.
Time-lapse imaging of Tg(foxd3:GFP) embryos was performed as previously described.23 (link) Briefly, the embryos were imaged using a TCS SP5 MP multiphoton microscope (Leica Microsystems CMS GmbH) fitted with a Mai Tai DeepSee Ti-Sapphire laser (Spectra-Physics; Newport Corp., Irvine, CA, USA). The images were obtained every 20 minutes during the described time frame. Each time-lapse experiment was repeated three or four times. Images and movies included are representative of all experiments.
4
Two-photon Calcium Imaging of Cortical Neurons
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To ensure consistent orientation of recordings, animals were aligned in the microscope such that the midline of the forebrain was parallel to the base of the field of view, with anterior to the right. Calcium imaging was performed using a Zeiss LSM 710 inverted two-photon microscope and Zen 2012 software (Carl Zeiss AG, Oberkochen). Samples were excited using a Spectra-Physics Mai TaiDeepSee TI:Sapphire laser (Spectra-Physics, CA) at an excitation wavelength of 940 nm. The emitted light was detected with a non descanned detector and a Plan Apochromat 10x/0.45 M27 objective (Carl Zeiss AG, Oberkochen). Time-lapse images were obtained from a cortical area of 870 × 650 μm for 10–30 mins at a sampling rate of 2.16 Hz and a pixel size of 2.18 μm.
5
Collagen Fiber Analysis in Mammary Glands
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Collagen fibers were imaged in picrosirius red-stained tissue sections from the fourth inguinal mammary glands of 12-week-old mice using an Olympus FV1000 MPE microscope equipped with a × 25 XLPlan objective (N.A. 1.05) and a Mai Tai DeepSee Ti:Sapphire Laser (Spectra-Physics, Newport Corp.) at a 950 nm wavelength. Images were taken through the depth of the sample in 4 μm sections, focusing on mammary ducts and the periductal area. A maximum intensity projection of these images was taken, and CT-Fire software (v1.3; https://loci.wisc.edu/software/ctfire ) was used to analyze the collagen fibers in each image. Three glands from oil-treated mice and 4 glands from BPA-treated mice were imaged, and 3 images were taken per tissue section. Overall, 467 collagen fibers in oil-treated mice and 1256 collagen fibers in BPA-treated mice were analyzed.
6
Zebrafish Larval Imaging via Two-Photon Microscopy
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Zebrafish larvae were embedded in 2.5% low-melting-point agarose, positioned at the centre of a 35 mm diameter plastic petri dish and overlaid with E3 embryo medium. Time-lapse two-photon images were acquired at the Queensland Brain Institute’s Advanced Microscopy Facility using a Zeiss LSM 710 inverted two-photon microscope. A custom-made inverter tube composed of a pair of beam-steering mirrors and two identical 60 mm focal length lenses arranged in a 4 f configuration was used to allow imaging with a 40x/1.0 NA water-dipping objective (Zeiss) in an upright configuration. Samples were excited via a Spectra-Physics Mai TaiDeepSee Ti:Sapphire laser (Spectra-Physics) at an excitation wavelength of 940 nm. Laser power at the sample plane ranged between 12 and 20 mW. Emitted light was bandpass filtered (500–550 nm) and detected with a nondescanned detector. Time-lapse images (416 × 300 pixels) were obtained at 2.2 Hz. To improve stability of the recording, chambers were left to settle prior to imaging for 3 h.
7
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.
8
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.
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.
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