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Sp8 2 photon microscope

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The Leica SP8 2-photon microscope is a high-performance imaging system designed for advanced research. It utilizes two-photon excitation technology to enable deep tissue imaging with minimal phototoxicity. The system's core function is to capture high-resolution, 3D images of fluorescently labeled samples.

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Lab products found in correlation

Hp deepsee laser, by Spectra-Physics (2 mentions) Ethyl cinnamate, by Merck Group (1 mentions) Phytagel, by Merck Group (1 mentions) Dylight 594 conjugated tomato lectin, by Vector Laboratories (1 mentions) Matlab, by MathWorks (1 mentions) Ti e inverted microscope, by Nikon (1 mentions) Evans blue, by Merck Group (1 mentions) Dichroic mirror, by IDEX Corporation (1 mentions) Mai tai hp deepsee laser, by Spectra-Physics (1 mentions)

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SP8 microscope (288 citations) SP8 Confocal Microscope with 2-Photon (2 citations) Leica TCS SP8 MP 2-photon intravital microscope (2 citations)

7 protocols using sp8 2 photon microscope

1

Visualizing Lymphatic Vessels with 2-Photon Microscopy

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Mesentery and mesLN were fixed and imaged using a customized Leica SP8 2-Photon microscope equipped with a 325/0.95 NA water-dipping objective and a Mai Tai HP DeepSee Laser (Spectra-Physics) tuned to 810 nm. Fluorescence emission was separated by 3 high-efficiency dichroic mirrors cutting at 458, 495, and 560 nm (Semrock) and directly directed to 4 supersensitive external detectors. 2-Photon excitation produced a second harmonic signal from collagen fibers within the analyzed tissue. Prox1-GFP reporter mouse was used to image lymphatic vessels.
Czepielewski R.S., Erlich E.C., Onufer E.J., Young S., Saunders B.T., Han Y.H., Wohltmann M., Wang P.L., Kim K.W., Kumar S., Hsieh C.S., Scallan J.P., Yang Y., Zinselmeyer B.H., Davis M.J, & Randolph G.J. (2021). Ileitis-associated tertiary lymphoid organs originate at lymphatic valves and obstruct mesenteric lymph flow in response to tumor necrosis factor. Immunity, 54(12), 2795-2811.e9.
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2

Imaging Neonatal Mouse Lungs

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Lungs from P7 neonatal mice treated with PBS or BMP9 were perfused with heparinized PBS before harvest and inflation via the trachea with 0.75% Phytagel (Sigma Aldrich). Lungs were then fixed overnight at 4 °C in 4% PFA and dehydrated by sequential overnight incubations in 50% ethanol at pH 9, 70% ethanol at pH 9, and 2 nights at 100% ethanol, respectively. Following dehydration, tissues were incubated overnight in ethyl cinnamate (Sigma Aldrich) for clarification before imaging on a Leica SP8 2-photon microscope.
Theilmann A.L., Hawke L.G., Hilton L.R., Whitford M.K., Cole D.V., Mackeil J.L., Dunham-Snary K.J., Mewburn J., James P.D., Maurice D.H., Archer S.L, & Ormiston M.L. (2020). Endothelial BMPR2 Loss Drives a Proliferative Response to BMP (Bone Morphogenetic Protein) 9 via Prolonged Canonical Signaling. Arteriosclerosis, Thrombosis, and Vascular Biology, 40(11), 2605-2618.
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3

Visualizing Mouse Lymphatic Dynamics

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The mouse hindlimb was treated with depilating cream and washed with PBS to remove hair. After removal of the superficial layer of the skin, lymphatic collectors were visualized via injection of 10 to 20 μl (s.c.) of DyLight 594–conjugated tomato lectin (Vector Laboratories) into the footpad. Images of popliteal lymphatics were collected using a customized Leica SP8 2-photon microscope equipped with a ×25/0.95 NA water-dipping objective. A Mai Tai HP DeepSee Laser (Spectra- Physics) tuned to 900 nm provided the excitation light. Fluorescence emission was separated by dichroic mirrors at 458, 495, and 560 nm (Semrock) and directed to external detectors. 2-Photon excitation produced a second harmonic signal from collagen. Vessel diameters were measured using Imaris (Bitplane) and Matlab (Mathworks). After the 594 nm channel was isolated in Imaris, a Matlab script was used to break the vessel image into 20 sections, and then the width was measured randomly within each section. The average value of all 20 measurements gave the average vessel diameter at each point in time. Graphs were generated from the average thickness at each point over the 1000 frames collected at 14 fps.
Davis M.J., Kim H.J., Zawieja S.D., Castorena-Gonzalez J.A., Gui P., Li M., Saunders B.T., Zinselmeyer B.H., Randolph G.J., Remedi M.S, & Nichols C.G. (2020). Kir6.1-dependent KATP channels in lymphatic smooth muscle and vessel dysfunction in mice with Kir6.1 gain-of-function. The Journal of physiology, 598(15), 3107-3127.
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4

Visualizing Fibrillar Collagen Deposition

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Fibroblasts were plated on 0.2% gelatin (crosslinked with 1% glutaraldehyde and 1 M ethanolamine)-coated plates (MatTek, P35G-1.5-14-C) and incubated overnight (day 0). Fresh medium supplemented with 75 μg ml–1 ascorbic acid was added on days 1, 3, 5 and 7. At day 8, cells were lysed carefully (0.5% Triton X-100 + 20 mM NH4OH), PBS was added and stored at 4 °C overnight to avoid disturbing the matrix. Matrix-coated plates were washed with PBS, and fresh PBS (supplemented with 100 U ml–1 penicillin and 100 mg ml–1 streptomycin) was added and sealed with Parafilm for up to 2–3 weeks at 4 °C. ECM deposition was first visualized using a Nikon TiE inverted microscope using phase-contrast optics. Fibrillar collagen deposition was detected using second-harmonic generation (SHG) microscopy using a Leica SP8 2-photon microscope with the laser tuned to a wavelength of 900 nm. The SHG ‘backwards scatter’ signal was imaged (five random fields of view per sample), and the fibrillar collagen area and intensity were quantified using NIS Elements software (v.4.60.00).
Verginadis I.I., Avgousti H., Monslow J., Skoufos G., Chinga F., Kim K., Leli N.M., Karagounis I.V., Bell B.I., Velalopoulou A., Salinas C.S., Wu V.S., Li Y., Ye J., Scott D.A., Osterman A.L., Sengupta A., Weljie A., Huang M., Zhang D., Fan Y., Radaelli E., Tobias J.W., Rambow F., Karras P., Marine J.C., Xu X., Hatzigeorgiou A.G., Ryeom S., Diehl J.A., Fuchs S.Y., Puré E, & Koumenis C. (2022). A stromal Integrated Stress Response activates perivascular cancer-associated fibroblasts to drive angiogenesis and tumour progression. Nature Cell Biology, 24(6), 940-953.
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5

Two-Photon Imaging of Cerebral Vasculature

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mTBI and control mice were imaged using a Leica SP8 2-photon microscope equipped with an 8000-Hz resonant scanner, a 25× collar-corrected water-dipping objective (1.0 NA), a quad HyD external detector array, and a Mai Tai HP DeepSee Laser (Spectra-Physics) tuned to 905 nm. Three-dimensional time-lapse movies were captured as Z stacks. For blood vessel visualization, 100 μL of 1 mg/mL Evans blue (MilliporeSigma) dissolved in PBS was injected i.v. immediately following surgical preparation. For all imaging, artificial cerebral spinal fluid (aCSF) (Harvard Apparatus, 597316) was used to submerge the lens above the thinned skull.
Mason H.D., Johnson A.M., Mihelson N.A., Mastorakos P, & McGavern D.B. (2021). Glia limitans superficialis oxidation and breakdown promote cortical cell death after repetitive head injury. JCI Insight, 6(19), e149229.
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6

Laser-Mediated Dendrite Ablation

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Larvae were mounted in 90% glycerol under No. one coverslips, dendrites were imaged using a Leica SP8 2-photon microscope with a 20 × 1.0 NA water immersion lens at 2x magnification under low (<20%) laser power. Cells were ablated or dendrites were severed by focusing high laser output (>80%) on the nucleus or a ~ 2 micron dendrite segment (64x magnification ROI scan), respectively. Larvae were recovered to yeasted agar plates with vented lids, aged at 25 ˚C, and processed for live imaging or immunostaining at the indicated time. Zebrafish axons were severed using a 2-photon laser as previously described (O'Brien et al., 2009b (link)).
Jiang N., Rasmussen J.P., Clanton J.A., Rosenberg M.F., Luedke K.P., Cronan M.R., Parker E.D., Kim H.J., Vaughan J.C., Sagasti A, & Parrish J.Z. (2019). A conserved morphogenetic mechanism for epidermal ensheathment of nociceptive sensory neurites. eLife, 8, e42455.
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7

In Vivo Adipose Tissue Imaging

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Mice were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) and maintained with halved doses administered every hour. The mouse lower abdomen was carefully shaved with Nair (Church & Dwight Co.) and washed with PBS to remove the excess lotion. A small incision was made in the lower abdomen in order to expose the eWAT pad which was kept moist with PBS and carefully positioned under a coverslip that was screwed into place for imaging. During the acquisition, mouse status was closely monitored. Images were collected using a customized Leica SP8 2-photon microscope equipped with a 25X/0.95 NA water-dipping objective and a Mai Tai HP DeepSee Laser (Spectra-Physics) tuned to 880 nm. Fluorescence emission was separated by 3 high-efficiency dichroic mirrors cutting at 458, 495, and 560 nm (Semrock) and directly directed to 4 supersensitive external detectors. These detectors and a resonant galvo scanner with 12,000 Hz frequency very short dwell times under relatively low laser power. This prevents photo-conversion of the Dendra2 protein. 3D stacks consisting of between 21 and 31 planes (0.5 μm step size) were captured every 30 seconds. Imaris software (BITPLANE, Inc.) was used to reconstruct 3-dimmensional (3-D) images, determine cellular localization with 3-D positional mapping, and generate movies derived from time-lapsed imaging.
Brestoff J.R., Wilen C.B., Moley J.R., Li Y., Zou W., Malvin N.P., Rowen M.N., Saunders B.T., Ma H., Mack M.R., Hykes BL J.r., Balce D.R., Orvedahl A., Williams J.W., Rohatgi N., Wang X., McAllaster M.R., Handley S.A., Kim B.S., Doench J.G., Zinselmeyer B.H., Diamond M.S., Virgin H.W., Gelman A.E, & Teitelbaum S.L. (2020). Intercellular mitochondria transfer to macrophages regulates white adipose tissue homeostasis and is impaired in obesity. Cell metabolism, 33(2), 270-282.e8.
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