To visualize migration of neutrophils, intravital imaging was performed using FV1000-AOM multiphoton system (Olympus) equipped with a 25x NA1.05 water immersion objective. For two-photon excitation, a Mai-Tai HP Ti:Sa Deep See laser system (Spectra-Physics) was tuned to 900nm for imaging. Neutrophils labeled with CellTracker™ Green CMFDA (Thermo Fisher Scientific Inc.) were intradermally injected into ear skin 2h before in vivo imaging. Mice were anesthetized by intraperitoneal injection of pentobarbital (Nembutal Sodium solution, Oak Pharmaceuticals, Inc., IL) and anesthetic condition was maintained using isoflurane. Hair of the mouse ear was removed using a commercial hair remover (Nair, Princeton, NJ). The anesthetized mice were laid in a ventral recumbent position on a custom-designed stage to expose the dorsal side of the ear pinna for imaging. Micropore™ (3M health care, MN) tapes were placed to immobilize the ear skin. Body temperature of the mice was controlled with heat pad and the ear immersed in a drop of glycerol/saline (40:60 v/v) and covered with a coverslip. Laser injury was induced by focusing the multiphoton laser tuned to 800 nm at a region within the ear dermis for 5 seconds. To track and analyze the movements of the neutrophils, Volocity software (Improvision/Perkin-Elmer, Waltham, MA) and ImageJ (National Institutes of Health, Bethesda, MD) were used.
Hp ti sa deep see laser system
Manufactured by Spectra-Physics
Sourced in United States
The HP Ti:Sa Deep See laser system is a high-performance titanium-sapphire laser. It delivers femtosecond pulses at a tunable wavelength range.
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3 protocols using hp ti sa deep see laser system
1
Intravital Imaging of Neutrophil Migration
2
Intravital Imaging of Neutrophil Migration
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To visualize migration of neutrophils, intravital imaging was performed using FV1000-AOM multiphoton system (Olympus) equipped with a 25x NA1.05 water immersion objective. For two-photon excitation, a Mai-Tai HP Ti:Sa Deep See laser system (Spectra-Physics) was tuned to 900nm for imaging. Neutrophils labeled with CellTracker™ Green CMFDA (Thermo Fisher Scientific Inc.) were intradermally injected into ear skin 2h before in vivo imaging. Mice were anesthetized by intraperitoneal injection of pentobarbital (Nembutal Sodium solution, Oak Pharmaceuticals, Inc., IL) and anesthetic condition was maintained using isoflurane. Hair of the mouse ear was removed using a commercial hair remover (Nair, Princeton, NJ). The anesthetized mice were laid in a ventral recumbent position on a custom-designed stage to expose the dorsal side of the ear pinna for imaging. Micropore™ (3M health care, MN) tapes were placed to immobilize the ear skin. Body temperature of the mice was controlled with heat pad and the ear immersed in a drop of glycerol/saline (40:60 v/v) and covered with a coverslip. Laser injury was induced by focusing the multiphoton laser tuned to 800 nm at a region within the ear dermis for 5 seconds. To track and analyze the movements of the neutrophils, Volocity software (Improvision/Perkin-Elmer, Waltham, MA) and ImageJ (National Institutes of Health, Bethesda, MD) were used.
3
Two-Photon Microscopy for Intravital Imaging
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A two-photon microscope LSM7MP (Carl-Zeiss, Oberkochen, Germany) was used to generate the imaging data. Zen 2012 blue edition software (Carl-Zeiss) was used for image acquisition and basic image analysis. Mai-Tai HP Ti:Sa Deep See laser system (Spectra-Physics, Mountain View, CA, USA) was used to generate an excitation laser (wavelengths of 880 nm, 810 nm, and 800 nm for intravital imaging with GFP/Texas red-dextran, three-dimensional cochlea imaging, and intravital imaging with FITC-dextran, respectively). The images were acquired at a resolution of 512 × 512 pixels using band-pass filters with wavelengths in the range of 420-480 nm (blue), 500-550 nm (green), and 575-610 nm (red). We used a 20× objective lens for all experiments.
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