Multiphoton imaging was done using a Zeiss LSM7 MP System equipped with a 20x/1.0NA water-immersion objective lens (Zeiss UK, Cambridge, UK) and a tunable Titanium: sapphire solid-state two-photon excitation source (Chamelon Ultra II; Coherent Laser Group, Glasgow, UK) and optical parametric oscillator (OPO; Coherent Laser Group). Popliteal LNs were excised 24 h after immunization, transferred into CO2-independent media at room temperature and bound onto a plastic coverslip with veterinary glue (Vetbond, 3M, St. Paul, MN). Grease was used to attach the coverslip to the bottom of the imaging chamber, which was supplied with warmed (36.5°C) and gassed (95% O2 and 5% CO2) RPMI 1640 before and during the imaging. A laser output of 820 nm and OPO signal at 1,060 nm provided excitation of YFP CD11c- and DS-Red OTII T cells. Acquisition of the videos was done for 20–30 min with X-Y pixel resolution of 512 × 512 inches in 2 μm. Cellular 3D tracking was done using Volocity 6.1.1 (Perkin Elmer, Cambridge, UK). Mean velocity, displacement and meandering index was calculated for each object. Intersection of DsREd and YFP objects was used to determine interaction between T cells and DCs, respectively (28 (link), 30 (link)).
Lsm7 mp system
Manufactured by Zeiss
Sourced in United Kingdom, Germany
The LSM7 MP system is a multiphoton laser scanning microscope designed for advanced imaging applications. It provides high-resolution, non-invasive imaging capabilities for a wide range of samples. The system's core function is to enable efficient and versatile multiphoton excitation and detection for various research and imaging needs.
Automatically generated - may contain errors
Lab products found in correlation
Volocity 6, by PerkinElmer (4 mentions)
Na water immersion objective lens, by Zeiss (4 mentions)
Gaasp photomultiplier tube, by Hamamatsu Photonics (4 mentions)
Vetbond, by 3M (3 mentions)
Mai tai hp, by Spectra-Physics (3 mentions)
Volocity 5, by PerkinElmer (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
Qdot 705, by Thermo Fisher Scientific (1 mentions)
Fv1000 mpe microscope, by Olympus (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
15 protocols using lsm7 mp system
1
Intravital Imaging of T Cell-DC Interactions
2
Intravital Imaging of T Cell-Dendritic Cell Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multiphoton imaging was carried out using a Zeiss LSM7 MP system equipped with a 20×/1.0 NA water-immersion objective lens (Zeiss UK, Cambridge, UK) and a tunable Titanium: sapphire solid-state two-photon excitation source (Chameleon Ultra II; Coherent Laser Group, Glasgow, UK) and optical parametric oscillator (OPO; Coherent Laser Group). Excised LNs were continuously bathed in warmed (37°C), oxygenated CO2 independent medium. A laser output of 820 nm and OPO signal at 1060 nm provided excitation of YFP CD11c+ and DsRed OT-II cells. Videos were acquired for 20 to 30 min with an X-Y pixel resolution of 512 × 512 in 2 μm Z increments. 3D tracking was performed using Volocity 6.1.1 (Perkin Elmer, Cambridge, UK). Values representing the mean velocity, displacement, and meandering index were calculated for each object. The intersection of DsRed and YFP objects was used to determine interaction between T cells and DCs respectively.
3
In Vivo Multiphoton Imaging of Mouse Popliteal
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Multiphoton imaging was performed with a Zeiss LSM7 MP system equipped with both a 10x/0.3 NA air and a 20x/1.0NA water immersion objective lens (Zeiss) and a tuneable titanium/sapphire solid state 2-photon excitation source (Chamelon Ultra II; Coherent Laser Group). For in vivo imaging, animals were anesthetized with 3% isoflurane in 1.5 L/min oxygen, anesthesia was maintained with the isoflurane at 1.5–2%, and oxygen at 1.5 L/min. Core body temperature was continuously monitored and maintained by a thermostatically controlled heat mat. The popliteal was surgically exposed and the leg fixed in place using surgical veterinary glue (Vetbond; 3M). The hind quarters of the mouse was submerged in PBS warmed and maintained at 35–37°C throughout the experiment (45 (link)). Videos were acquired for 15–30 min at an X-Y pixel resolution 512 × 512 with 2 μm increments in Z. Images were processed using Volocity 5.5 (Perkin Elmer) after correction for tissue drift.
4
Neuron Morphometry Using Microscopy and Image Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After patch-clamp recording and filling with biocytin/neurobiotin tracer, horizontal midbrain slices were fixed in 4% paraformaldehyde (PFA) solution for 24 hr at 4°C. All following staining procedures were done as described previously (Marx et al., 2012 (link); Mao et al., 2019 (link)), depending on the tracer. Biocytin-filled, DAB (3,3′-Diaminobenzidine) stained neurons (N = 27) were imaged with Zeiss Axio Imager Z2 microscope (Zeiss AG, Oberkochen, Germany) in transmitted light mode. Neurobiotin-filled, Streptavidin-Alexa 633 stained neurons (n = 22) were imaged with multiphoton Zeiss LSM 7 MP system. 3D morphology reconstruction was done with Simple Neurite Tracer plugin (https://imagej.net/Simple_Neurite_Tracer ) in Image J software, followed by inbuilt Sholl analysis instrument.
5
Intravital Multiphoton Microscopy for In Vivo Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intravital multi-photon microscopy studies were carried out using a Zeiss LSM7 MP system equipped with a tuneable titanium:sapphire solid-state two-photon excitation source (4W, Chameleon Ultra II, Coherent Laser Group) coupled to an Optical Parametric Oscillator (Chameleon Compact OPO; Coherent). Movies were acquired for 10 to 15 min with an X:Y pixel resolution of 512 × 512 in 2 μm Z increments producing up to 40 μm stacks. 3D tracking was performed using Volocity 6.1.1 (Perkin Elmer, Cambridge, UK). Values representing the mean velocity, displacement and meandering index were calculated for each object. Mice were anaesthetised IP using medetomidine (Domitor 0.5 mg/kg) and ketamine (50 mg/kg) and placed on a heated stage. Following removal of hair with a depilatory cream, dorsal skin was imaged. An intravenous injection of non-targeted quantum dots (Qdot705) (Life Technologies, UK) prior to imaging allowed visualisation of blood vessels.
6
In vivo calcium imaging of cortical neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ca2+ imaging of L2/3 pyramidal neurons was conducted using a LSM 7 MP system (Zeiss) and a mode-locked Ti:sapphire Chameleon Ultra II laser (Coherent) tuned to 950 nm, with a 20× objective (XLPlan, NA of 1.0; Zeiss). Fluorescence was separated by a 570-nm dichroic mirror with 495–550 nm (green channel: for EGFP fluorescence detection) and 570–630 nm (red channel: for tdTomato fluorescence detection) emission filters and was collected using GaAsP photomultiplier tubes (Hamamatsu Photonics). The laser intensity was 5–30 mW. The imaged fields were 424.27 × 424.27 μm, at a depth of 150–200 μm below the cortical surface. The pixel size was 0.830 μm. Frame duration was 390 ms, and continuous 1000-frame imaging was repeated for each field for 10–30 min. For imaging the apical dendrites (and their spines) of L5 pyramidal neurons in awake mice, the image plane was focused at a depth within 50 μm from the cortical surface (L1). The image fields were 28.28 × 28.28 μm, with a pixel size of 0.055 μm and a frame duration of 242 ms. Continuous 3000-frame imaging was repeated in each field for 12 min.
7
Two-Photon Imaging of Lever-Pull Task
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two‐photon images were acquired from the left M1 during performance of the lever‐pull task (on Days 1, 3, 5, 7, and 9) using either an FV1000‐MPE microscope (Olympus, Tokyo, Japan) with a ×25 objective (XLPlan, NA 1.05; Olympus) and a mode‐locked Ti:sapphire laser (MaTai HP; Spectra Physics, CA) at a wavelength of 910–920 nm, or an LSM 7 MP system (Zeiss, Jena, Germany) with a ×20 objective (XLPlan, NA 1.0; Zeiss) and a mode‐locked Ti:sapphire Chameleon Ultra II laser (Coherent, Santa Clara, CA) tuned to 920 nm. Fluorescence was collected with GaAsP photomultiplier tubes (Hamamatsu Photonics, Shizuoka, Japan). Laser intensity was 20–40 mW. The imaged fields were 508.93 × 508.93 μm at 150–200 μm depth below the cortical surface. Pixel size was 1.988 μm. Frame duration was 420 ms. Continuous 1,000‐frame imaging was repeated in each field for 10–30 min. The lever position and solenoid signal were recorded simultaneously with the Ca2+ imaging. Two‐photon imaging in WT/PV‐Cre and PLP‐tg/PV‐Cre mice was performed on Day 1.
8
Intravital Imaging of Neutrophil Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lyz2-GFP+ mice (males, 10 weeks old) were anesthetized by ketamine/xylene injection and placed on the imaging stage at 37°C. Mice were injected in the ear pinna with 10 ng of IL-1β (R&D Systems #401ML-005) with or without the CD31agonist (50 µM). Mice were transferred immediately to the microscope stage and imaged for 15–20 min for each time point at an X-Y pixel resolution of 512 × 512. Multiphoton imaging was performed with a Zeiss LSM7 MP system equipped with both a ×10/0.3 NA air and ×20/1.0 NA water-immersion objective lens (Zeiss) and a tunable titanium/sapphire solid-state two-photon excitation source (Chameleon Ultra II; Coherent Laser Group; Stephen et al., 2017 (link)). Videos were processed offline with the ImageJ software for the generation of time-superposition images and the GFP+ morphological parameters; the TrackMate plugin was implemented to calculate the speed of detaching neutrophils by applying a region of interest out of the vessel wall. TrackMate was also used to calculate the migratory path and the velocity of interstitial GFPhigh neutrophils. Data are from two independent experiments in which one mouse was used per condition.
9
Intraluminal Salmonella Infection Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eight-week-old female C57BL/6 mice were euthanized by cervical dislocation, and sections of colon were ligated and then immediately removed and maintained in warmed Hanks balanced salt solution (HBSS) (Gibco). Approximately 5 × 107 SL1344, ΔSipA, or ΔSipA/pSipA-phiLOV cells were injected intraluminally along with Image-iT Live Red caspase-3/-7 substrate (Molecular Probes). Multiphoton imaging through the intestinal wall was carried out using a Zeiss LSM7 MP system equipped with a 20×/1.0 NA water immersion objective lens (Zeiss UK, Cambridge, UK) and a tunable titanium-sapphire solid-state two-photon excitation source (Chameleon Ultra II; Coherent Laser Group, Glasgow, UK) and optical parametric oscillator (OPO) (Coherent Laser Group). Images were reconstructed postmicroscopy into 3D images using ZEN software (Zeiss).
10
Two-Photon Imaging of FRET Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Z-stacks of explant regions with high numbers of MNTs were acquired using a Zeiss LSM7 MP system, using a 20×/1.0NA water-immersion objective lens and a tuneable titanium/sapphire solid-state 2-photon excitation source (Chameleon Ultra II; Coherent Laser Group), tuned to 840 nm. Light was first filtered through a 685-nm LP dichroic. For FRET imaging, an LSM binary GaAsP photodetector module was used with CFP/YFP filterset cube (BP 455–500/BP 525–570) with an LP510 dichroic mirror.
After NHS incubation, tissue was rapidly rinsed 3×with Ringer’s and images were taken between 11 and 12 min after NHS was initially added. To minimise imaging time and prevent photodamage to the tissue, images were taken with 1.2–1.3 μm z-slice and pixel size of 754 nm with 2× line averaging.
After NHS incubation, tissue was rapidly rinsed 3×with Ringer’s and images were taken between 11 and 12 min after NHS was initially added. To minimise imaging time and prevent photodamage to the tissue, images were taken with 1.2–1.3 μm z-slice and pixel size of 754 nm with 2× line averaging.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!