Tcs sp8 multiphoton confocal microscope

Manufactured by Leica

Sourced in Germany

The TCS SP8 multiphoton confocal microscope is a high-performance imaging system designed for advanced fluorescence microscopy applications. It features a laser-scanning technology that allows for the acquisition of high-resolution, three-dimensional images of biological samples. The instrument is equipped with a multiphoton excitation capability, enabling the visualization of deep tissue structures with improved penetration depth and reduced phototoxicity compared to conventional confocal microscopy.

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Lab products found in correlation

H 3300, by Vector Laboratories (2 mentions) 4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions) Axioplan 2 microscope, by Zeiss (1 mentions) 4 6 diamino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions) Topro 3, by Thermo Fisher Scientific (1 mentions) Fluoromount g medium, by Electron Microscopy Sciences (1 mentions) Imagej, by Oxford Instruments (1 mentions)

Close spelling variants of this product

TCS SP8 multiphoton microscope Leica SP8 confocal/multiphoton microscope TCS-SP8 multiphoton SP8 multiphoton microscope Confocal TCS SP8 TCS SP8 microscope SP8 confocal microscope SP8 multiphoton TCS SP8 Confocal SP8

6 protocols using tcs sp8 multiphoton confocal microscope

Multimodal Imaging of Chromosome Dynamics

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Imaging was performed on a Leica TCS SP8 Multiphoton Confocal microscope using a 1.3 NA ×40 oil immersion objective with pixels of 541.6 nm × 541.6 nm. Fields of view were selected, such that confluence was balanced to provide maximum data points and to ensure proper cell segmentation during downstream analysis. Z-stacks were determined, such that both homologs were within the imaged space and represented 10 μm in total axial thickness. Localizations were then recorded in 0.3 μm steps.
Representative images were also obtained on a Leica TCS SP8 Multiphoton Confocal microscope, this time using a 1.4 NA ×63 oil immersion objective with pixels of 343.9 × 343.9 nm. Z-stacks were determined and recorded as with the 1.3 NA ×40 oil immersion objective. Each cell, allele, and locus for each strain were individually processed using FIJI software to generate orthogonal projections of the Z-stacks and to split channels into individual TIFs.
3D reconstructions of representative cells were rendered using IMARIS v.7.4.2 software (Bitplane AG, Switzerland). DNA FISH dots were generated using the Spots tool with a 0.8 μm diameter, created at the intensity mass center of the fluorescent probe signal. Nuclear volume was created using the Surfaces tool with automatic settings based on the fluorescent signal from the DAPI stain.

Wang W., Chandra A., Goldman N., Yoon S., Ferrari E.K., Nguyen S.C., Joyce E.F, & Vahedi G. (2022). TCF-1 promotes chromatin interactions across topologically associating domains in T cell progenitors. Nature immunology, 23(7), 1052-1062.

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Immunofluorescence Assay for H2A.X Phosphorylation and Nrf2 Localization

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For immunofluorescence experiments assessing H2A.X phosphorylation on Ser139 and Nrf2 localization, skin fibroblasts were cultured on glass coverslips before treatment and they were then fixed with 4% (w/v) formaldehyde in PBS. Labeling was performed as previously described [31 (link), 37 (link)] using antibodies against phospho-H2A.X (Ser139) and Nrf2 and a FITC-conjugated IgG. Nuclear staining was performed with 2 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride (Sigma). Labeled cells were visualized using a Zeiss AxioPlan 2 microscope (Carl Zeiss, Jena, Germany) and a confocal laser scanning microscope (TCS SP8 multiphoton confocal microscope, Leica, Mannheim, Germany).

Mavrogonatou E., Angelopoulou M., Rizou S.V., Pratsinis H., Gorgoulis V.G, & Kletsas D. (2022). Activation of the JNKs/ATM-p53 axis is indispensable for the cytoprotection of dermal fibroblasts exposed to UVB radiation. Cell Death & Disease, 13(7), 647.

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Collagen Fiber Analysis in Mammary Tumors

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Mouse mammary tumor tissues were preserved at −80°C with optimal cutting temperature compound. Samples were transferred to −20°C for at least 2 hours and cut into 20 µm thick sections with a cryostat. Slides were then incubated at 37°C for 30 min and transferred to boiling antigen unmasking solution (catalog no. H-3300, Vector labs) for 10 min. Samples were nuclear stained with To-pro-3 (catalog no. T3605, Thermo Scientific) and mounted onto coverslips (No.1.5) by adding Fluoromount-G mounting media (catalog no. 100502-406, VWR). Images were acquired with a Leica TCS SP8 multiphoton confocal microscope and a 20×, HC PL Apo, NA 0.7 oil-immersion objective was used throughout the experiments. Tuned excitation wavelength was 840 nm. A 420±5 nm narrow bandpass emission controlled by a slit was used to detect the second harmonic generation (SHG) signal of collagen.29 (link) Collagen fiber assessment was analyzed with CT Fire software (V.2.0 beta) (https://loci.wisc.edu/software/ctfire). The area on the tumor side with 400–600 µm distance from the tumor-stroma border was defined as tumor margin.

Liu J., Chiang H.C., Xiong W., Laurent V., Griffiths S.C., Dülfer J., Deng H., Sun X., Yin Y.W., Li W., Audoly L.P., An Z., Schürpf T., Li R, & Zhang N. (2023). A highly selective humanized DDR1 mAb reverses immune exclusion by disrupting collagen fiber alignment in breast cancer. Journal for Immunotherapy of Cancer, 11(6), e006720.

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Immunofluorescence Staining of Senescent Cells

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For the immunofluorescence staining of the lipofuscin-containing senescent cells [43 (link)] with SenTraGor (Lab Supplies Scientific, Athens, Greece), human primary breast stromal fibroblasts were plated on glass coverslips and fixed with 4% (v/v) formaldehyde in PBS. Staining was performed according to the manufacturer’s instructions. In brief, coverslips were washed with TBS, followed by a wash with 50% (v/v) ethanol and a wash with 70% (v/v) ethanol. Samples were then incubated with SenTraGor reagent (the chemical compound GL13 linked with biotin) until detection of the signal under a light microscope. After removal of the excess SenTraGor reagent, samples were washed with 50% (v/v) ethanol and TBS, before incubation with a phycoerythrin-conjugated anti-biotin antibody (Invitrogen) and counterstained with 2 μg/mL 4′,6-diamino-2-phenylindole (DAPI) dihydrochloride (Sigma). Samples were visualized under a confocal laser scanning microscope (TCS SP8 multiphoton confocal microscope, Leica, Mannheim, Germany).

Mavrogonatou E., Papadopoulou A., Fotopoulou A., Tsimelis S., Bassiony H., Yiacoumettis A.M., Panagiotou P.N., Pratsinis H, & Kletsas D. (2021). Down-Regulation of the Proteoglycan Decorin Fills in the Tumor-Promoting Phenotype of Ionizing Radiation-Induced Senescent Human Breast Stromal Fibroblasts. Cancers, 13(8), 1987.

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Quantifying Collagen in Mammary Tumor Tissue

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Mouse mammary tumour tissue was embedded and preserved in optimal cutting temperature (OCT) compound at −80 °C. Before cutting, samples were brought to −20 °C for at least 2 h and a 20-μm-thick section was cut using a cryostat. Slides were thawed and incubated at 37 °C for 30 min and then transferred to boiling antigen unmasking solution (Vector labs, H-3300) for 10 min. After nuclear staining with To-pro-3 (Fisher, T3605), each tumour section was mounted with fluoromount-G medium (Electron Microscopy Science, 17984–25) onto a microscope coverslip (no. 1.5). All samples were imaged using a Leica TCS SP8 multiphoton confocal microscope and a 20×, HC PL Apo, NA 0.7 oil-immersion objective was used throughout the experiments. The excitation wavelength was tuned to 840 nm^{34 (link)}, and a 420 ± 5 nm narrow bandpass emission controlled by a slit was used for detecting the SHG signal of collagen. SHG signal is generated when two photons of incident light interact with the noncentrosymmetric structure of collagen fibres, which leads to the resulting photons being half the wavelength of the incident photons. Collagen measurement was performed using CT-Fire software (v.2.0 beta) (https://loci.wisc.edu/software/ctfire). Tumour margin for SHG analysis is defined as an area on the tumour side with a depth of 60 μm from the tumour–stroma border.

Sun X., Wu B., Chiang H.C., Deng H., Zhang X., Xiong W., Liu J., Rozeboom A.M., Harris B.T., Blommaert E., Gomez A., Garcia R.E., Zhou Y., Mitra P., Prevost M., Zhang D., Banik D., Isaacs C., Berry D., Lai C., Chaldekas K., Latham P.S., Brantner C.A., Popratiloff A., Jin V.X., Zhang N., Hu Y., Pujana M.A., Curiel T.J., An Z, & Li R. (2021). Tumour DDR1 promotes collagen fibre alignment to instigate immune exclusion. Nature, 599(7886), 673-678.

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Quantitative Multimodal Imaging of BACE1

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Single- or double-immunolabeled (Alexa Fluor-488, -568 or -647) samples were analyzed at the Imaging & Molecular Biology Platform (IMBP; McGill Life Sciences Complex) using a TCS SP8 multi-photon confocal microscope (Leica) with 63x/1.40 oil-immersion objectives (Leica, Wetzlar, Germany). Samples were excited with Coherent Chameleon Vision II multiphoton at 730 nm (2660 mW) for DAPI imaging. For each sample, 12–30 z-stack images were acquired using the same laser intensity settings for quantification. Z-stack images were processed using Image-J (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997–2018) and total cell fluorescence was quantified with the analyze tool. To better visualize BACE1 localization, a heatmap was generated using Fire LUT in Image-J. The IMARIS Image Analysis Software (Bitplane (Oxford Instruments), MA, USA) software was used for cross-sectional analysis. BACE1 colocalization with EEA1 and LAMP1 were analyzed using ImageJ plugin JACoP⁶⁶.

Ulku I., Liebsch F., Akerman S.C., Schulz J.F., Kulic L., Hock C., Pietrzik C., Di Spiezio A., Thinakaran G., Saftig P, & Multhaup G. (2023). Mechanisms of amyloid-β34 generation indicate a pivotal role for BACE1 in amyloid homeostasis. Scientific Reports, 13, 2216.

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