SpectraSplit® 7

Bandpass filters for 7-color multiplex IHC

SpectraSplit 7 are bandpass filters tailor made for 7-color multiplex immunohistochemistry (IHC). The filter sets efficiently separate, without computational spectral unmixing, seven classes of fluorochromes that are frequently used for multiplex IHC including, including OpalTM dyes and Alexa Fluor® dyes and traditional dyes, such as FITC, Texas redTM, Cy3®, Cy5®, Cy7® (table 1). The fluorochrome specificity also entails high signal-to-noise ratios (figure 1), which is of extra importance when visualizing tissues with weak immunostainings and/or tissues with high autofluorescence. 

Filter set configuration and dimensions

SpectraSplit 7 consists of four filter sets that are compatible with standard and scanning fluorescence microscopes. Each filter set contains one excitation filter, one emission filter (25 mm in diameters), and one dichroic mirror (25.5 x 36.0 x 1 mm, Figure 2). Set 1: Triple-band set (DAPI/488/750), Set 2: Double-band set (440/647), Set 3: Single-band set (Cy3), Set 4: Single-band set (594). 

Figure 1. Multistained frozen tissue section from mouse spleen visualized with SpectraSplit 7. Antibodies and dyes: DAPI, anti-CD11c (tyramide CF430), anti-CD4 (tyramide CF488), anti-CD45R (tyramide Cy3), anti-CD8a (tyramide CF594), anti-F4/80 (tyramide Cy5), and anti-Ly6C/G (tyramide Opal polaris 780). 

Fluorochrome chart

SpectraSplit 7 is compatible with a wide range of common fluorochromes (table 1). Thus, fluorochromes of the same or of different brands can be used in a 7-color multiplex staining without spectral overlap during imaging.  No post processing is necessary – what you see is what you get! 

Table 1. Examples of dyes that are specifically detected by each individual SpectraSplit7 channel.

Opal dyes are trademark of Akoya Biosciences, Alexa Fluor and DyLight dyes are trademarks of Thermo Fisher Scientific, CF dyes are trademarks of Biotium, Atto dyes are trademarks of Atto-tec, CL dyes are trademarks of Cell IDx

Light sources

SpectraSplit 7 is compatible with pE-800 and pE-4000 from CoolLED, X-Cite NOVEM from Excelitas (remove clean-up filters in NOVEM for best result), and SPECTRA III Light Engine from Lumencor. Other light sources have not been tested.