Hyd sp

Manufactured by Leica

The HyD SP is a hybrid detector designed for sensitive and efficient detection of fluorescence signals in microscopy applications. It combines the advantages of photomultiplier tubes and silicon photon detectors to provide high quantum efficiency and low noise performance.

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Lab products found in correlation

Sp5 x mp multiphoton microscope, by Leica (1 mentions) Las x software, by Leica (1 mentions)

3 protocols using hyd sp

Multiphoton Microscopy of GFP-Expressing Cells

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Immunostained tissues were imaged using an inverted Leica SP5-X MP multiphoton Leica microscope connected to a Ti-Sapphire laser (Spectra Physics MaiTai HP DeepSee Laser), Spectral Physics (tunable wavelength: 690 –1040 nm). The objective used was a HCX PL APO lambda blue 63 × 1.20 NA WATER UV. Tissue sections containing GFP-expressing cells were stained for expression of SCRG1 (Alexa 555) and Nidogen-2 (Alexa 647). Fibrillar collagen was imaged by means of second harmonic generation (SHG) using two-photon excitation at 892 nm and emission between 426 and 446 nm was detected using a hybrid detector (HyD SP, Leica). GFP-expressing cells were simultaneously excited using the 892 nm two-photon excitation and emitted GFP-light was collected between 505 and 550 nm using a PMT detector. SCRG1 (Alexa 555) and Nidogen-2 (Alexa 647) was excited with a supercontinuum white light laser (WLL) and emitted light (Alexa 555: 567–612 nm and Alexa 647: 650–710 nm) were detected using the hybrid detector (HyD SP, Leica). All images are from back-scattered light and captured with a resolution of 1024 × 1024 pixels, at 200 Hz.

Bartoschek M., Oskolkov N., Bocci M., Lövrot J., Larsson C., Sommarin M., Madsen C.D., Lindgren D., Pekar G., Karlsson G., Ringnér M., Bergh J., Björklund Å, & Pietras K. (2018). Spatially and functionally distinct subclasses of breast cancer-associated fibroblasts revealed by single cell RNA sequencing. Nature Communications, 9, 5150.

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Super-resolution Imaging of Fluorescent Samples

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Immunofluorescence stainings for g-STED imaging were performed as described for conventional confocal microscopy, yet fluorescently labeled secondary Abs were diluted 1:100. Leica TCS SP8 g-STED 3× laser scanning microscope was used to acquire super-resolved images (Leica Microsystems). A Leica STED HC PL APO 100×/1.40 objective was used. Fluorochromes and fluorescent proteins were excited at the optimal wavelength by means of 80 MHz pulsed white light laser (470–670 nm), allowing time gating of fluorescence lifetimes. For STED, the appropriate, 592 or 660 nm, depletion laser was used. 592 and 660 nm depletion lasers were used at 40% and 70% of their nominal power. Fluorescence channels were scanned sequentially, and emission was revealed by means of hybrid spectral detectors (HyD SP; Leica Microsystems). Time-gated detection was also used, and detection was delayed by 0.5 ns. Images were analyzed using ImageJ quantification tool.

Mana G., Valdembri D., Askari J.A., Li Z., Caswell P., Zhu C., Humphries M.J., Ballestrem C, & Serini G. (2022). The βI domain promotes active β1 integrin clustering into mature adhesion sites. Life Science Alliance, 6(2), e202201388.

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Quantifying CIK Cell-Spheroid Interactions

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GFP⁺ STS spheroids were co-cultured with CSPG4-CAR.CIK or unmodified NTD.CIK cells stained with red dye PKH26 at E:T ratio 2:1 in culture medium (300 U/mL IL-2 at 37°C, 5% CO₂). Following a 16 hour co-culture at 37°C, CIK cells were removed and immunofluorescence acquisition was conducted on the remaining spheroids. Briefly, STS spheroids were washed twice, and then centrifuged at 300 g for 3 min in PBS, fixed in paraformaldehyde 4% for 1 hour, resuspended with mounting medium, and applied on either glass slides or glass bottom chamber slide wells. STS spheroids were observed using a Leica SP8 AOBS confocal microscope. Next, 80MHz pulsed white light laser (470-670 nm) was used to excite the fluorochromes in the spheroids. Fluorescence channels were scanned sequentially, and hybrid spectral detectors (HyD SP Leica Microsystems) revealed the emissions. Image acquisition of the STS spheroids was performed maintaining the same laser power, gain, offset, and magnification (20x). We generated maximum intensity projections for each analysed spheroid with LAS X software (Leica) to quantify CIK cell recruitment and infiltration. Images of the total PKH26 red fluorescent area (μm²) present either at the boundary or inside the spheroid were analysed using ImageJ software.

Leuci V., Donini C., Grignani G., Rotolo R., Mesiano G., Fiorino E., Gammaitoni L., D’Ambrosio L., Merlini A., Landoni E., Medico E., Capellero S., Giraudo L., Cattaneo G., Iaia I., Pignochino Y., Basiricò M., Vigna E., Pisacane A., Fagioli F., Ferrone S., Aglietta M., Dotti G, & Sangiolo D. (2020). CSPG4-specific CAR.CIK lymphocytes as a novel therapy for the treatment of multiple soft tissue sarcoma histotypes. Clinical cancer research : an official journal of the American Association for Cancer Research, 26(23), 6321-6334.

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