Thy1-YFP and Thy1-YFP:5XFAD mice were injected with Alexa594-conjugated fibrinogen (Invitrogen) intravenously daily for 3 days as described15 (link). Mice were also injected i.p. with Methoxy-X04 24 h prior to imaging. Methoxy-X04 was solubilized with DMSO:propylene glycol:PBS pH 7.5 (ratio 2:9:9) at 5 mg/ml. On day of imaging, a small craniotomy was made and a custom-made metal plate affixed to a stage to stabilize the skull. Alexa594-fibrinogen solution was injected retro-orbitally prior to imaging. The anesthetized animal was placed on a heated pad under an Ultima-IV multiphoton microscope (Prairie) equipped with MaiTai DeepSee-eHP lasers (Spectra Physics). The excitation wavelength was 820nm to simultaneously visualize fibrinogen, methoxy-X04 and YFP dendrites. Imaging was performed from 20 to 150 μm below the dura, using a Nikon 40 × 0.8 NA -immersion lenses with a 1.0 μm z-step. z-stacks of images were projected along the z-axis to recreate two-dimensional representations of the 3D structures within the imaged volumes. Images were adjusted for brightness, contrast and background noise with ImageJ. Spectral unmixing plugin in ImageJ was used to separate overlapping signals.
Maitai deepsee ehp lasers
Manufactured by Spectra-Physics
The MaiTai DeepSee-eHP laser is a mode-locked Ti:Sapphire laser that provides high-energy ultrashort pulses. It features a compact design and delivers excellent beam quality and stability.
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Lab products found in correlation
3 protocols using maitai deepsee ehp lasers
1
Imaging Alzheimer's Disease Pathology
2
Imaging Alzheimer's Disease Pathology
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Thy1-YFP and Thy1-YFP:5XFAD mice were injected with Alexa594-conjugated fibrinogen (Invitrogen) intravenously daily for 3 days as described15 (link). Mice were also injected i.p. with Methoxy-X04 24 h prior to imaging. Methoxy-X04 was solubilized with DMSO:propylene glycol:PBS pH 7.5 (ratio 2:9:9) at 5 mg/ml. On day of imaging, a small craniotomy was made and a custom-made metal plate affixed to a stage to stabilize the skull. Alexa594-fibrinogen solution was injected retro-orbitally prior to imaging. The anesthetized animal was placed on a heated pad under an Ultima-IV multiphoton microscope (Prairie) equipped with MaiTai DeepSee-eHP lasers (Spectra Physics). The excitation wavelength was 820nm to simultaneously visualize fibrinogen, methoxy-X04 and YFP dendrites. Imaging was performed from 20 to 150 μm below the dura, using a Nikon 40 × 0.8 NA -immersion lenses with a 1.0 μm z-step. z-stacks of images were projected along the z-axis to recreate two-dimensional representations of the 3D structures within the imaged volumes. Images were adjusted for brightness, contrast and background noise with ImageJ. Spectral unmixing plugin in ImageJ was used to separate overlapping signals.
3
Intravital Imaging of Microglia in Mouse Cortex
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For intravital imaging with two-photon microscopy, thinned-skull windows were made in 12-17-monthold Cx3cr1 GFP/+ hTREM R47H/+ , Cx3cr1 GFP/+ hTREM2 CV/+ , and Cx3cr1 GFP/+ mTrem2 +/+ as described previously 36 . Briefly, mice were anesthetized, the skull was exposed, and a small area over the cortex was thinned manually and with a high-speed drill (K.1070 High Speed Rotary Micromotor drill; Foredom). Mice were fixed onto a custom-made head plate and imaged with an Ultima IV multiphoton microscope (Bruker) equipped with MaiTai DeepSee-eHP lasers (Spectra Physics) tuned to 920 nm for imaging and InSight X3 lasers (Spectra Physics) tuned to 880 nm for ablation. Z-stacks of images were acquired every 3 min in 1-µm steps with a 25x water-immersion objective at 2.4 optical zoom.
Extensions and retractions of processes during baseline recordings were manually traced with the mTrackJ plugin. The movement of microglial cells toward a laser ablation site was analyzed by normalizing the number of processes near the injury site at each time point to the overall microglial density at that time point.
Extensions and retractions of processes during baseline recordings were manually traced with the mTrackJ plugin. The movement of microglial cells toward a laser ablation site was analyzed by normalizing the number of processes near the injury site at each time point to the overall microglial density at that time point.
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