In vivo imaging of dendritic spines through the cranial window was performed on a two-photon microscope (Ultima Investigator, Bruker Co., Middleton, WI, USA) using a 16x/0.8 NA water-immersion objective (Nikon Instruments, Inc., Melville, NY, USA) and an ultrafast two-photon laser (Mai Tai, Spectra-Physics, Santa Clara, CA, USA) operating at 940 nm wavelength. The mouse was anesthetized with a mixture of ketamine (20 mg/ml) and xylazine (2.0 mg/ml) in 0.9% sterile saline administered intraperitoneally (5 ml/kg bodyweight). It was then placed onto a custom-made holding stage, secured by the head-bar. Prior to the first imaging session, images of blood vessels were taken under a dissection microscope as a reference for subsequent relocations. Stacks of two-photon images were taken at 12x zoom with a z-step size of 1 μm. After the first imaging session, low-magnification image stacks (1x and 4x zoom, z-step size = 3 μm) were taken to facilitate relocation.
Ultima investigator
Manufactured by Bruker
Sourced in United States
The Ultima Investigator is a high-performance analytical instrument designed for materials science research and development. It utilizes advanced X-ray diffraction (XRD) technology to provide comprehensive analysis of crystalline and polycrystalline materials. The instrument enables accurate phase identification, structural characterization, and quantitative analysis of a wide range of samples.
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Lab products found in correlation
8 protocols using ultima investigator
1
In vivo Imaging of Dendritic Spines
2
Somatic Calcium Imaging in Rabbits and Monkeys
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Neuronal structure and somatic calcium imaging in rabbits was performed using a two-photon microscope (Bruker Ultima Investigator equipped with TI: Sapphire Laser) with the laser tuned to 920 nm and with a 20X air objective (N.A. 0.5). A 2 X digital zoom was used to yield images (295 × 295 μm, 512 × 512 pixels) for the quantification of neuronal structure (GFP/td-Tomato labeled) and calcium transients. With the Bruker's two-photon imaging system, the laser power and pixels dwell time for imaging were ~ 30 mw and 1.6 µs, respectively.
Neuronal structure and somatic calcium imaging in monkeys were performed using a Thorlabs Multiphoton Imaging Microscope (Bergamo II, Thorlabs Imaging Systems) with the laser tuned to 920 nm and with a 10X air objective (N.A. 0.5). A 3X digital zoom was used to yield images (443 × 443 μm, 256 × 256 pixels) for quantification of somatic structure (td-Tomato labeled) and calcium activity. With the Thorlabs' multiphoton imaging system, the laser power and pixels dwell time for imaging were ~ 30 mw and 5 µs, respectively.
Neuronal structure and somatic calcium imaging in monkeys were performed using a Thorlabs Multiphoton Imaging Microscope (Bergamo II, Thorlabs Imaging Systems) with the laser tuned to 920 nm and with a 10X air objective (N.A. 0.5). A 3X digital zoom was used to yield images (443 × 443 μm, 256 × 256 pixels) for quantification of somatic structure (td-Tomato labeled) and calcium activity. With the Thorlabs' multiphoton imaging system, the laser power and pixels dwell time for imaging were ~ 30 mw and 5 µs, respectively.
3
Two-Photon Imaging of Cortical Neuronal Activity
4
Two-Photon Imaging of Cortical Layer 2/3
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Images were acquired using a resonant scanning two-photon microscope (Ultima Investigator, Bruker, WI) at a 30 Hz frame rate and 512 × 512 pixel resolution through a 16x water immersion lens. PPC was imaged at a depth between 150 and 250 μm, at the level cortical layer 2/3. The angle of the objective was matched to the angle of the window. Excitation light was provided by both a tunable femtosecond infrared source (780–1100 nm) and a fixed 1045 nm wavelength laser (Insight X3, Spectra-Physics, CA). Tunable and fixed wavelength beams were combined with a dichroic mirror (ZT1040dcrb-UF3, Chroma, VT) before being routed to the microscope’s galvanometers. Note that because of this optics configuration, imaging cannot be performed at tunable wavelengths immediately surrounding 1045 nm. Green and red wavelengths were separated through a 565 nm lowpass filter before passing through bandpass filters (Chroma, ET525/70 and ET 595/50, VT). PrairieView software (vX5.5 Bruker, WI) was used to control the microscope.
5
Multimodal Imaging of Vascular Networks
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A two-photon microscopy system (Bruker Ultima Investigator) equipped with a laser source (InSight DeepSee, 680–1300 nm and 1040 nm, Spectra Physics) was employed to perform ex vivo imaging of samples. Two-photon excitation of methoxy-X04 (10 mg/kg, i.p., Abcam ab142818, USA) and DyLight649 labeled lectin from Lycopersicon esculentum (Vector DL-1178) was performed at 810 nm and each emission signal was separately detected by two GaAsp detectors and digitally recorded for image analysis. Methoxy X04 was i.p. injected 24 h prior to the imaging. Mice under anesthesia received an i.v. injection of DyLight649 labeled lectin through the jugular vein 5 min prior to sacrifice for ex vivo imaging. Tissues including the brain, ileum, and cecum were collected followed by sample fixation in 4% PFA. After a couple of washes with 1 xpbs, samples were mounted inside a 7 cm petri dish with 1× pbs. The meningeal vasculature in the parietal cortex was imaged from the pial surface of the region, hippocampal vasculature was imaged in the sagittally mounted brain tissues, ileum and cecum were imaged from luminal surface of each tissues cut and mounted to expose the lumen. All regions were imaged 2–3 times per tissue and Vision 4 D software (Arivis, DC, USA) was used to analyze the image data.
6
Two-Photon Imaging of Neural Dynamics
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The optical setup for two-photon imaging was composed of a pulsed laser source (Chameleon Ultra, 80 MHz repetition rate tuned at 920 nm, Coherent) and Bruker Ultima Investigator equipped with 6 mm raster scanning galvanometers, movable objective mount, 16x/0.8 NA objective (CFI75 LWD 16X W, Nikon, Milan), and multi-alkali photomultiplier tubes. Laser beam intensity was adjusted using a Pockel cell (Conoptics Inc, Danbury). Laser beam power at the objective outlet was 90–110 mW. GCaMP6f or TdTomato emission signal was collected by the photomultipliers after band-pass filtering (525/70 nm) and digitalized in 12 bits. Imaging sessions were conducted in raster scanning mode. t-series were motion corrected using an open-source implementation of up-sampled phase cross-correlation 71 (link), 80 (link) and the t-series median projection was used as reference frame. One or two weeks after surgery the animals were handled by the operator for a minimum of two days and habituated to the imaging setup. Starting from the second session, the animals were head-restrained for a progressively increasing amount of time, reaching 1 hour in approximately one week. Mice were free to run on a custom 3D printed wheel. Experimental sessions lasted approximately one hour. After each session, animals were returned to their home cages.
7
Quantifying Actin Alignment and Nuclear Morphology
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Two-Photon Imaging of Aversive Shock Response
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