SpectraSplit® 7

SpectraSplit® 7 are patented filter sets that empower fluorescence microscopes with seven color channels.  The channels are distributed over four filter sets and specifically separate the most commonly used fluorochromes in immunofluorescence (see table below). Hence, you can immunostain tissues with up to seven fluorochromes and still visualize each fluorochrome in the sample with SpectraSplit® 7 without bleedthrough. No additional software and no computational spectral unmixing are required.

SpectraSplit® 7 can easily be installed in both standard and scanning fluorescence microscopes, and can be run with different popular light sources. See the description below for more information.

Cat no. 7001991-1

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Description

The SpectraSplit® 7 filter sets are highly efficient in separating the emission signals of seven different fluorochrome classes, with spillover between channels being less than 0.5%. As a result, high-contrast images are generated without the need for spectral unmixing or any other bleed-through corrections.

SpectraSplit® 7 immediately empowers your microscope with seven independent color channels that each generates crystal clear images. Importantly, you do not need to be a fluorochrome expert or microscope guru to produce 7-color.

Fluorochromes
SpectraSplit® 7 is fully compatible with commonly used fluorochromes, such as AlexaFluor® dyes, as well as the classic FITC/Cy3/Cy5/Cy7 dyes and many of the OpalTM dyes.

Fluorochromes compatible with SpectraSplit® 7

Channel Traditional dyes StreptaClick® dyes ThermoFisher Biotium ThermoFisher Atto-tec Akoya Ultivue Sirigen/BD Protein
expression
fluorophores
S-Split Blue (375) DAPI AZDye™ 405 Alexa Fluor 405 CF405 Dylight 405 DAPI DAPI BV421 Hoecht
S-Split Cyan (435) CF®430 CF430 Atto425 Opal p.480 BV480 cCFP
S-Split Green (490) FITC AZDye™ 488 Alexa Fluor 488 CF488 DyLight 488 Atto488 Opal 520 Ultivue, FITC eGFP
S-Split Orange (545) Cy3/TRITC AZDye™ 555,
Cyanine 3, Atto™ 542
Alexa Fluor 546 CF555 DyLight 549 Atto542 Opal 570 Ultivue, Cy3 mOrange/
mRFP
S-Split Red (590) Texas Red AZDye™ 594 Alexa Fluor 594 CF594 DyLight 594 Atto590 Opal 620 mCherry/mRaspberry
/mPlum
S-Split Far-red (650) Cy5/Cy5.5 AZDye™ 647,
Cyanine 5
Alexa Fluor 647 CF647/680 DyLight 649 Atto647/665 Opal 690 Ultivue, Cy5 miRFP703
S-Split Infra-red (740) Cy7 Alexa Fluor 750 CF750 DyLight 750 Atto740 Opal p.780 Ultivue, Cy7

*Cy3, Cy5, Cy7 are registered trademarks of GE Healthcare, Alexa Fluor and DyLight dyes are trademarks of Thermo Fisher Scientific, CF dyes are trademarks of Biotium, Atto dyes are trademarks of Atto-tec, Opal dyes are trademarks of Akoya Biosciences, Brillialt Violet dyes are trademarks of Sirigen/BD

Bandwidth
The approximate mid excitations for the different channels are at 375, 435, 490, 545, 590, 650, and 740nm. However, the exact bandwidths are proprietary information and not publicly disclosed.

Microscopes and light sources
SpectraSplit® 7 is designed to work with both standard and scanning fluorescence microscopes. It is compatible with various lighting sources including pE-800 and pE-4000 from CoolLED, X-Cite NOVEM from Excelitas, and the new SPECTRA X Light Engine from Lumencor.

Hardware
SpectraSplit® 7 contains four custom made filter sets, manufactured to the high product standards of Chroma Technologies. SpectraSplit® 7 is a patented technology, exclusive to our brand. These filter sets are not available under any other name. Each filter set includes one excitation filter, one emission filter (25 mm in diameter), and one dichroic mirror (25.5 x 36.0 x 1 mm).

Set 1: Triple-band set (375/490/740)
Set 2: Double-band set (435/650)
Set 3: Single-band set (545)
Set 4: Single-band set (590)

Downloads

FOLDER
SpectraSplit® 7 folder

Publications

PUBLICATIONS

Bonstingl, L., Zinnegger, M., Sallinger, K. et al. Advanced single-cell and spatial analysis with high-multiplex characterization of circulating tumor cells and tumor tissue in prostate cancer: Unveiling resistance mechanisms with the CoDuCo in situ assay. Biomark Res 12, 140 (2024). https://doi.org/10.1186/s40364-024-00680-z

Gribonika, I. et al. Migratory CD103+CD11b+ cDC2s in Peyer’s patches are critical for gut IgA responses following oral immunization. Mucosal Immunology, Volume 17, Issue 4, 509 - 523, https://doi.org/10.1016/j.mucimm.2024.03.004

Rodin, W., Szeponik, L., Rangelova, T. et al. γδ T cells in human colon adenocarcinomas comprise mainly Vδ1, Vδ2, and Vδ3 cells with distinct phenotype and function. Cancer Immunol Immunother 73, 174 (2024). https://doi.org/10.1007/s00262-024-03758-7

[PREPRINT] Bonstingl, L. et al. Circulating tumor cell characterization and classification by novel combinatorial dual-color (CoDuCo) in situ hybridization and supervised machine learning, bioRxiv 2024.05.08.592946; https://doi.org/10.1101/2024.05.08.592946

Andric, F.; Al-Fairouzi, A.; Wettergren, Y.; Szeponik, L.; Bexe-Lindskog, E.; Cusack, J.C., Jr.; Tumusiime, G.; Quiding-Järbrink, M.; Ljungman, D. Immune Microenvironment in Sporadic Early-Onset versus Average-Onset Colorectal Cancer. Cancers 2023, 15, 1457. https://doi.org/10.3390/cancers15051457

Liang, F.; Nilsson, L.M.; Byvald, F.; Rezapour, A.; Taflin, H.; Nilsson, J.A.; Yrlid, U. A Fraction of CD8+ T Cells from Colorectal Liver Metastases Preferentially Repopulate Autologous Patient-Derived Xenograft Tumors as Tissue-Resident Memory T Cells. Cancers 2022, 14, 2882. https://doi.org/10.3390/cancers14122882

Caër C, Gorreja F, Forsskåhl SK, Brynjolfsson SF, Szeponik L, Magnusson MK, Börjesson LG, Block M, Bexe-Lindskog E, Wick MJ. TREM-1+ Macrophages Define a Pathogenic Cell Subset in the Intestine of Crohn's Disease Patients. J Crohns Colitis. 2021 Aug 2;15(8):1346-1361. https://doi.org/10.1093/ecco-jcc/jjab022

Glise L, Rutberg M, Håversen L, Levin MC, Levin M, Jeppsson A, Borén J, Fogelstrand P. pH-Dependent Protonation of Histidine Residues Is Critical for Electrostatic Binding of Low-Density Lipoproteins to Human Coronary Arteries. Arterioscler Thromb Vasc Biol. 2022 Aug;42(8):1037-1047. doi: 10.1161/ATVBAHA.122.317868. Epub 2022 Jun 2. PMID: 35652335. https://doi.org/10.1161/ATVBAHA.122.317868

Alsén S, Cervin J, Deng Y, Szeponik L, Wenzel UA, Karlsson J, Cucak H, Livingston M, Bryder D, Lu Q, Johansson-Lindbom B and Yrlid U (2022) Antigen-Presenting B Cells Program the Efferent Lymph T Helper Cell Response. Front. Immunol. 13:813203. https://doi.org/ 10.3389/fimmu.2022.813203

Liang, F.; Rezapour, A.; Szeponik, L.; Alsén, S.; Wettergren, Y.; Bexe Lindskog, E.; Quiding-Järbrink, M.; Yrlid, U. Antigen Presenting Cells from Tumor and Colon of Colorectal Cancer Patients Are Distinct in Activation and Functional Status, but Comparably Responsive to Activated T Cells. Cancers 2021, 13, 5247. https://doi.org/10.3390/cancers13205247

Håversen, L., Sundelin, J.P., Mardinoglu, A. et al. Vimentin deficiency in macrophages induces increased oxidative stress and vascular inflammation but attenuates atherosclerosis in mice. Sci Rep 8, 16973 (2018). https://doi.org/10.1038/s41598-018-34659-2

Yrlid U, Holm M, Levin M, Alsén S, Lindbom M, Glise L, Bergh N, Borén J, Fogelstrand P. Endothelial repair is dependent on CD11c+ leukocytes to establish regrowing endothelial sheets with high cellular density. J Leukoc Biol. 2019 Jan;105(1):195-202. doi: 10.1002/JLB.4A1017-402RR. Epub 2018 Sep 28. PMID: 30265749. https://doi.org/10.1002/JLB.4A1017-402RR

S. Kijani, A. M. Vázquez, M. Levin, J. Borén, P. Fogelstrand. Intimal hyperplasia induced by vascular intervention causes lipoprotein retention and accelerated atherosclerosis, Physiol Rep, 5 (14), 2017, e13334, https://doi.org/10.14814/phy2.13334

** Kijani S, Yrlid U, Heyden M, Levin M, Borén J, Fogelstrand P (2015) Filter-Dense Multicolor Microscopy. PLoS ONE 10(3): e0119499. https://doi.org/10.1371/journal.pone.0119499

** The original publication demonstrating what is now our SpectraSplit® bandpass filters for multiplex immunofluorescence microscopy.

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