Ai6 mice were immunized i.p. with 105 CPS-Cre-OVA-mCherry parasites, and 18 hours later 2 × 105 naïve, CTV-labeled OT-I/Nur77GFP T cells and 2 × 105 naïve, CTY-labeled P14 T cells were transferred via i.p. injection. Starting 2 hours later, recipient Ai6 mice were anesthetized with isoflurane and maintained at a core temperature of 37°C. The peritoneum was surgically opened to access the omentum, and a small portion of visceral adipose tissue was resected to assist in immobilizing the omentum via a soft-tissue vacuum apparatus (VueBio). No greater than 40 kPa of pressure was applied to steady the omentum for imaging. Image acquisition was performed on a Leica SP8 multiphoton confocal with a 20X water immersion objective (1.0 NA) with resonant scanner (8000 kHz) and 4 external HyD detectors. The excitation wavelength of Chameleon Vision II Ti:Sapphire laser was tuned for optimal detection of the labeled OT-I and P14 T cells in each experiment, typically 880 nm. Images collected x-, y-, and z-plane data over time with a step size of 2 μm and a z-thickness that allowed for a complete z-series every 22s. This was carried out for approximately 30 minutes for each region imaged. Resulting images were segmented in Imaris (v9.7.2, Bitplane) with spot-specific position data exported and analyzed in R with the CellTrackR package.
Sp8 multiphoton
Manufactured by Leica
The Leica SP8 multiphoton is a microscope system designed for advanced imaging techniques. It utilizes multiphoton excitation to capture images and data from specimens. The core function of the SP8 multiphoton is to provide high-resolution, non-invasive imaging capabilities for a variety of biological and materials science applications.
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5 protocols using sp8 multiphoton
1
Intravital Imaging of T Cell Responses to Parasite Infection
2
Intravital Imaging of Lymphatic Flow
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Inert Fluoresbrite YG carboxylate nanospheres (50 nm; Polysciences) at 0.025% in 10 μl PBS were injected into the left inguinal lymph node of anaesthetized 8-week-old female BALB/c nu/nu mice. Visualization of nanospheres was carried out using a Leica SP8 multiphoton as described above. Emission was collected at 455/25 (second harmonic generation), 525/25 (nanospheres) and 585/20 nm (mCherry tumour cells). The baseline rate of flow of nanospheres through the efferent lymphatic vessel was quantified after 3 min had elapsed to allow flow to stabilize. To investigate stress signalling in anaesthetized mice, stress neurotransmitter norepinephrine (Sigma; 1 mg kg−1) was injected intravenously and impact on lymph flow investigated. To avoid confounding of anatomical variation, lymph flow readings were taken in each mouse before and after norepinephrine administration. Quantification of lymphatic flow rate was carried out by manually tracking individual particles using the Fiji distribution of ImageJ over ⩾10 time points. The average velocity of each particle was then calculated in μm s−1.
3
In Vivo Imaging of Omental CD8+ T Cells
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Mice were anesthetized with isofluorane and maintained at a core temperature of 37°C. The peritoneum was opened minimally to access omentum. A small portion of visceral adipose tissue was resected to assist in immobilizing the omentum via a soft-tissue vacuum apparatus (VueBio). No greater than 40 kPa of pressure was applied to steady the omentum for imaging. Image acquisition was performed on a Leica SP8 multiphoton confocal with a 20× water immersion objective (1.0 NA) with a resonant scanner (8,000 kHz) and four external HyD detectors. The excitation wavelength of Chameleon Vision II Ti:Sapphire laser was tuned for optimal detection of CellTrace dye–labeled CD8+ T cells in each experiment, typically 880 nm. Images collected x-, y-, and z-plane data over time with a step size of 2 μm and a z-thickness that allowed for a complete z-series every 22 s. This was carried out for ∼30 min for each region imaged. The resulting images were segmented in Imaris (v9.7.2; Bitplane) with spot-specific position data exported and analyzed in R with the CellTrackR package (Wortel et al., 2021 (link)).
4
Visualizing Skin Inflammation with Multiphoton
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Skin was removed at various time points after injury and fixed in 4% paraformaldehyde for 4 hours. Skins were permeabilized and blocked using a buffer containing 0.1% TritonX-100. Samples were blocked with 1% BSA and incubated with Ly6G antibody conjugated to Alexa Fluor 647 overnight at 4°C. Skins were mounted on glass slides, and Kimwipes soaked in sterile PBS were used to keep the samples from drying. Leica SP8 multiphoton was used to visualize collagen fibers.
5
Live/Dead Cell Quantification via Calcein/PI
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Gels were incubated 1h in medium supplemented with calcein AM 2 µM and propidium iodide 6.6 µg/ml, transferred to fresh medium for washing and imaged on a Leica SP8 multiphoton over 465x465x200 µm. Live and dead cells were counted manually on the maximum intensity projection (MIP) (typically ~400 cells/image). Experiments were reproduced in triplicate with 3 different litters (n=9).
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