A two-photon microscope with a tunable Mai Tai laser (100 fs, 80 MHz; Spectra-physics, Santa Clara, CA) for excitation and a modified Olympus Fluoview 300 confocal unit was used for imaging. The scheme of the imaging setup is shown in Figure S2 . An Olympus water immersion objective lens LUMPlan fI/IR 20X/0.95NA and a C-Apochromat 10X/0.45 (Zeiss) were used to image bone in vivo via Second Harmonic Generation (SHG) and blood vessels via tdTomato Red fluorescence (RFP) of labeled endothelial cells in Cdh5-CreERT2; Ai9 mice. Images (1024×1024 pixels) were acquired at a rate corresponding to the pixel dwell time of 0.2 ms with the laser tuned to 810nm for SHG and 900nm for RFP, respectively. The autofluorescence of PCL fibers, fluorescence of RFP and SHG were collected with a 534/30 nm, a 605/55 nm and a 405/30 nm bandpass filters (Semrock), respectively.
Mai tai laser
Manufactured by Spectra-Physics
Sourced in United States, Germany
The Mai Tai laser is a widely-used ultrafast laser system from Spectra-Physics. It is a mode-locked titanium-sapphire laser that generates ultrashort pulses in the near-infrared spectrum. The core function of the Mai Tai laser is to provide a reliable source of high-quality, tunable femtosecond pulses for a variety of scientific and industrial applications.
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Lab products found in correlation
Fluoview 300 confocal unit, by Olympus (2 mentions)
Xlumplanfl, by Olympus (2 mentions)
Slicescope pro 6000, by Spectra-Physics (2 mentions)
568 hydrazide, by Thermo Fisher Scientific (2 mentions)
A1r confocal microscope, by Nikon (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Multiphoton trimscope 1 system, by Miltenyi Biotec (2 mentions)
C apochromat 10x 0, by Zeiss (1 mentions)
Fitc dextran, by Merck Group (1 mentions)
Lsm 710 microscope, by Zeiss (1 mentions)
Water immersion objective lens, by Olympus (1 mentions)
Fv1000 system, by Olympus (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Xlfluor 4 340, by Olympus (1 mentions)
Oregon green bapta 1, by Thermo Fisher Scientific (1 mentions)
Fluo 5f, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Ix51w, by Olympus (1 mentions)
Fluoview 300, by Olympus (1 mentions)
Fv300 microscope, by Olympus (1 mentions)
24 protocols using mai tai laser
1
In vivo Bone and Vasculature Imaging
2
Chronic Imaging of Mouse Visual Cortex
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Chronic imaging of mouse visual cortex was performed using a thinned skull
preparation as previously described [21 ], using GFP-M mice [22 (link)] that received 100 mg/L of fluoxetine in drinking water for
4 weeks. Briefly, a two-photon microscope with a Mai Tai laser (Spectra Physics)
and a modified Olympus Fluoview 300 confocal unit was used. An Olympus LUMPlan
fI/IR 20X/0.95NA was used to identify the binocular visual cortex based on
cortical vasculature; an area containing brightly labeled neurons was chosen for
imaging. 3D image stacks were obtained at high magnification to allow for
dendritic spine reconstruction in layers 1 and 2 of the visual cortex. After the
initial imaging session, the scalp was sutured and the animals were returned to
the animal facility. The animals were re-anesthetized 4 days later and the same
area was identified based on the blood vessel and dendritic patterns [21 ]. 3D image stacks of the same dendritic
regions were again obtained at high magnification. The percentage of lost and new
spines was determined relative to the total number of spines present in the
initial imaging session using ImageJ.
preparation as previously described [21 ], using GFP-M mice [22 (link)] that received 100 mg/L of fluoxetine in drinking water for
4 weeks. Briefly, a two-photon microscope with a Mai Tai laser (Spectra Physics)
and a modified Olympus Fluoview 300 confocal unit was used. An Olympus LUMPlan
fI/IR 20X/0.95NA was used to identify the binocular visual cortex based on
cortical vasculature; an area containing brightly labeled neurons was chosen for
imaging. 3D image stacks were obtained at high magnification to allow for
dendritic spine reconstruction in layers 1 and 2 of the visual cortex. After the
initial imaging session, the scalp was sutured and the animals were returned to
the animal facility. The animals were re-anesthetized 4 days later and the same
area was identified based on the blood vessel and dendritic patterns [21 ]. 3D image stacks of the same dendritic
regions were again obtained at high magnification. The percentage of lost and new
spines was determined relative to the total number of spines present in the
initial imaging session using ImageJ.
3
Intravital Multiphoton Imaging of Metastasis
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Metastasis formation was followed by in vivo-microscopy (Figure 1 B). For this purpose, a TrimScope multiphoton microscopy platform (LaVision Biotech TrimScope I) equipped with a MaiTai-laser (wavelength 690-1040nm; Spectra Physics, Newport) and a 20-times water immersion objective (numerical aperture 0.95; XLUMPlanFl, Olympus) was used. 6 standardized regions of interest (ROIs) per animal were longitudinally assessed with a x20 objective. Anesthesia during microscopy was established with 1% to 2% isoflurane in oxygen adjusted to the breathing rate; and the mice's head was fixated in a custom-made holding device. Subsequently, 0.2mL fluorescein isothiocyanate (FITC)-dextran (10mg/mL, 2 MDa molecular mass; Sigma-Aldrich) were injected into the tail vein for intravascular plasma staining. Cortical vessels were identified by the fluorescent signal of FITC-dextran and served as landmarks to retrieve the same ROIs during repetitive imaging. Image stacks with x/y/z-dimensions of 450 × 450 × 300 μm were acquired at a wavelength of 920nm. Imaging started at the brain surface (as defined by detection of the arachnoid fibers using second harmonic imaging) and recordings were made every 2µm. Image resolution was set at 1024 × 1024 pixels.
4
Two-Photon Microscopy for Formulations
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Formulations were imaged using a tuneable titanium sapphire Mai Tai laser (80 MHz, <200 fs, Spectra Physics, Mountain View) capable of two-photon excitation 710–910 nm. A spectral emission scan was performed on a Zeiss LSM 710 microscope (Carl Zeiss Microscopy GmbH, Berlin, Germany). Zen Black 2010 version 1.1.2.0 (Carl Zeiss Microscopy GmbH, Berlin, Germany) was used to extract intensity data, before manual correction for caprylic capric triglyceride (CCT) (Dow Chemical Company, Frenchs Forest, Australia) as background control and normalisation for laser power.
5
Two-Photon Calcium Imaging in Brain Slices
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After the last Miniscope imaging recording, some mice were sacrificed and brain slices containing cerebral cortex and striatum (350 μm) were obtained for electrophysiological recordings and Ca2+ imaging in vitro using 2PLSM. Ca2+ imaging was performed using a Scientifica 2PLSM equipped with MaiTai laser (Spectra-physics). To image Ca2+ signals, the laser was tuned at 920 nm wavelength. The laser power measured at the sample was less than 30 mW with a 40 × water-immersion objective lens (Olympus, Tokyo, Japan). Ca2+ transients were sampled at a rate of 3.81 Hz using SciScan software (Scientifica, Uckfield, UK). Optical signal amplitudes were expressed as ΔF/F, where F represents the resting light intensity at the beginning of the optical trace, and ΔF represents the change in peak fluorescence during the biological signal. These values were then multiplied by 100 to obtain percentage values. ImageJ (NIH) was utilized to process raw fluorescence values, which were then exported to Excel (Microsoft, Redmond, WA, USA) and Clampfit software to calculate % ΔF/F.
6
Optical pH Imaging of Astroglial pH Dynamics
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Slices were incubated in aCSF containing BCECF (5 µM) for 45 min followed by a through washing out to allow de-esterification of the dye. Optical recordings of changes in pHi were performed in a flow-through imaging chamber mounted on a stage of an Olympus FV1000 system optically linked to a Ti:Sapphire MaiTai laser with λ2P = 820 nm (Spectra Physics). Recordings were performed at ~33−35 °C in aCSF saturated with 95% O2/5% CO2 (pH 7.4; 300–310 mOsmol). Astroglial loading with BCECF was confirmed by analysing the characteristic morphology of the BCECF-stained cells featuring astrocytes, with no fluorescence detected in principal neurons (CA1–CA3 pyramidal neurons) (Fig. 2b ). For time-lapse recordings of pHi changes, images of BCECF-loaded astrocytes were collected using 256 × 256-pixel frames in the stream acquisition mode. Changes in pHi are expressed as changes in BCECF fluorescence at the maximum of the fluorescent signal over the baseline (ΔF/F0). Control optical recordings were performed in the same time frame without Schaffer fibre stimulation applied.
7
Fluorescent Microscopy of LEDGF Transfection
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Cells were transfected using Lipofectamine 2000 (Life Technologies, Merelbeke, Belgium) as described earlier [62 (link)]. LEDGF-hybrids were detected with the primary polyclonal rabbit anti-LEDGF480-530 antibody (A300-848a; 1/500; Bethyl Laboratories-Imtec Diagnostics N.V., Antwerpen, Belgium) and secondary polyclonal goat anti-rabbit antibody (1/500 in PBS, goat-αRb488; Bethyl Laboratories-Imtec Diagnostics N.V., Antwerpen, Belgium). Confocal images were acquired using an LSM 510 META imaging unit (Carl Zeiss, Zaventem, Belgium). Alexa-488 was excited at 488 nm (AI laser), mRFP at 543 (HeNe laser) and DAPI at 790 nm (Spectra-physics Mai Tai laser; Spectra Physics, Mountain View CA). After the main beam splitter (HFT KP 700/543 for mRFP, HFT UV/488/543/633 for eGFP, and HFT KP650 for DAPI) a secondary dichroic beam splitter was used to divide the fluorescence signal (NFT 490 for eGFP, NFT 545 for mRFP). Distinct signals were directed to different detectors and data analysis was performed with the LSM image browser. Overlay images were obtained using ImageJ freeware.
8
Multiphoton Imaging with Customized Microscope
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Multiphoton imaging was performed using a custom-built, laser scanning multiphoton microscope as described in refs. 39 (link),41 (link). In a subset of experiments, the galvanometric scanners were replaced with a resonant scanning system consisting of an 8 kHz resonant scanner on one scan axis (Sutter Instruments, CA, USA) and conventional galvanometric scanner on the other. Excitation was provided by a Mai Tai laser (Spectra Physics, CA, USA) at 920 nm and images acquired using Scanimage software (Vidrio Technologies, VA, USA). The objective lens used for all functional imaging was a Zeiss ×20 1.0 NA objective (Carl Zeiss AG, Oberkochen, Germany). In experiments using the conventional galvanometric scanning system, 128 × 64 pixel full frame images were acquired at a frame rate of 18.6 Hz, and in those using the resonant scanning system, 512 × 512 pixel full frame images were acquired at 29.98 Hz.
9
Multimodal Microscopy for Neuronal Morphology
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Two microscopes were used to visualize cell morphology. The in vitro microscope, a Scientifica SliceScope Pro 6000, used 980 nm illumination from a SpectraPhysics MaiTai Laser steered by a galvo scanner for 2P excitation and IR visualization. Software was SciScan version 1.5 by Scientifica in LabVIEW by National Instruments. Dyes for two-photon visualization were Alexa Fluor 488 and 568 Hydrazide from Invitrogen (A10436, A10437), the latter of which was found to be not gap junction permeable and was used for single-cell image isolation. Microscopy was continued on fixed retinas, which were stained with antibodies and fluorescent dyes for immunohistochemistry. The fixed-tissue microscope was a Nikon A1R confocal microscope with a 1.0 NA 40x oil immersion objective at the Northwestern Center for Advanced Microscopy. In Fig 1a –c and 7a , neurons were traced using Simple Neurite Tracer47 in ImageJ/Fiji. Stratification analysis in Fig. 1c used ChAT layers to computationally flatten traced neural morphology. In Fig. 4 individual image channels from confocal microscopy were contrast adjusted and despeckled with a 3×3 median filter to improve clarity, then max-intensity projected through 7 μm of depth.
10
Multiphoton Imaging of Cerebral Vasculature
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For TPLSM, a Multiphoton TrimScope I system (LaVision BioTec) connected to an upright Olympus microscope equipped with a MaiTai Laser (690 to 1,040 nm; Spectra Physics) and a 20× water immersion objective (numerical aperture [NA] 0.95; Olympus XLUMPlanFl) or a 4× objective (NA 0.28; Olympus XLFluor 4×/340) was used. During microscopy, mice were anesthetized using isoflurane in oxygen at a concentration of 1.0 to 2.0% adjusted according to breathing rate. During imaging, the PEEK ring was tightly secured into a custom-built fixation device. For visualization of cerebral vessels, 0.1 mL fluorescein isothiocyanate (FITC)-dextran (2 MDa molecular mass) was injected i.v. at a concentration of 10 mg/mL.
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