Advancing Cellular Analysis With Spectral Flow Cytometry

Since then, we have developed a new generation of NovoCyte cytometers, based on silicon-photomultipliers (SiPMs), with enhanced detection sensitivity while maintaining a smaller footprint. These include the 4 laser NovoCyte Quanteon (in 2018) with up to 25 fluorescent channels, 1–3 laser NovoCyte Advanteon (in 2019) with up to 21 fluorescent channels and 5 laser NovoCyte Penteon (in 2020) with 30 fluorescent channels.

To complement these changes, in 2023, we launched the new generation NovoExpress software, enabling CFR21 part 11 compliance for enhancing customers’ productivity and efficiency, particularly in the manufacturing environment.

Flow cytometry measures cells through fluorescent-labeled antibodies bound to the cell surface or intracellular proteins at a single-cell resolution. For a conventional cytometer, each antibody with corresponding fluorochrome is detected by one fluorescent channel. For example, a NovoCyte Quanteon with 25 detection channels can measure up to 25 antibodies for detecting 25 proteins or biomarkers for individual cells.

In a spectral cytometer, the combined fluorescent light from all the fluorochromes bound to a single cell is detected across the entire fluorescent emission spectra. Through spectral unmixing, one can resolve the fluorescent contribution from each of the fluorochromes. Spectral flow cytometers can provide users with better capabilities to differentiate and detect fluorochromes with similar emission profiles, to remove autofluorescence and ultimately to increase antibody/fluorochrome panel sizes for deeper biological insights.

We developed Opteon as a complete flow analyzer portfolio, to offer customers advanced spectral flow capabilities while retaining NovoCyte’s legacy of great performance, high reliability and ease of use.

a) The proprietary optical sub-system for fluorescent light collection, splitting and detection incorporates a total of 73 optical detectors and up to 5 lasers, while maintaining a bench-top instrument footprint.  

b) Avalanche photodiodes, a highly sensitive semiconductor photodiode that converts light to electricity, are employed in NovoCyte Opteon for better spectral measurement especially for an accurate generation of reference-control spectra at different gain settings. 

c) The optical sub-system works in synergy with stable flow control for both sheath and sample fluids at high and low sampling rates and, together with advanced, high-performance analog-to-digital conversion circuits, ensures an optimized and highly reproducible fluorescence collection and measurement.

d) A proprietary signal processing algorithm for noise reduction and signal enhancement allows for an electronic signal output at 24-bit resolution.  

e) Furthermore, onboard temperature control and electronic and fluidic sensor circuitry for real-time monitoring of instrument status ensures consistent and reliable data acquisitions under different ambient environments and varied high-and-low sampling rates.

f) Innovative spectral unmixing algorithms were developed and implemented capable of working with different reference-control setups to derive the relative contributions from individual fluorochromes.  

NovoExpress software has a built-in fluorochrome library (937 fluorochromes covering all major commercially available fluorochromes), each of which can be readily found and added to the assay panel, including basic info such as fluorochrome name, excitation wavelength and max emission wavelength. With the application of Avalanche photodiodes for fluorescence light detection providing a linear signal-to-gain relationship, NovoCyte Opteon offers flexible options for generating reference-controls (from single color-stained cell samples or single color-stained bead samples) obtained at different gain settings even at different times from those when a spectral flow experiment was conducted. An unmixing workflow is available for multiple reference-control options and unstained samples to check which reference-controls work best.

Cellular autofluorescence emitted by cellular endogenous molecules (such as NADH, flavins and lipofuscin) can mask the fluorescence signals from antibody-labeled fluorochromes, especially in larger and more granular cells. Spectral flow cytometry treats autofluorescence as an additional color, allowing it to be removed through spectral unmixing. This process enhances the resolution of low-expressing targets and dim levels of fluorescence signal. NovoExpress offers a versatile autofluorescence (AF) subtraction capability, providing an easy workflow for the exploration of different AF populations for spectral unmixing.

In addition, through the incorporation and use of virtual filters for each fluorochrome molecule, NovoExpress software further offers capabilities of data analysis with conventional compensation matrices that users are already familiar with.

Other powerful functionalities offered by NovoExpress software include similarity index matrix, spillover spreading matrix and modified cross stain index, significantly simplifying panel design optimization. Spectral unmixing can be achieved simultaneously during data acquisition. Easy-to-use compensation adjustment tools post-unmixing further help users to refine spectral unmixing and improve data quality. Powerful NxN plots, displaying all possible pairs of two fluorochromes in dot plots, make it easy to visualize and review the unmixing results and data quality.  

Finally, fluorescent photodetector gains can be adjusted individually, or per laser line, or all together to accommodate signal intensity with a large panel. 

For immunology research, a large, complex antibody panel is needed for a comprehensive study of different types of immune cells, as well as their functional status, such as activation, exhaustion, differentiation and maturation. In some cases, primary cell samples may exhibit high auto-fluorescence, where spectral cytometer Opteon could provide better resolution and sensitivity for low expression biomarkers, compared with a conventional non-spectra flow.

For cell therapy development, a relatively large size of biomarker panel is needed, whether the flow assay is for an individual (autologous therapy) or donor (allogenic therapy) immune cell characterization, or for cell therapy analytics development (development and validation of in-production-process cell assays for monitoring and qualifying cell therapy production process). Spectral flow may have the unique benefit of relative ease and capability of potentially adding one or more biomarkers to an already developed panel, meeting the individual sample needs, required in personalized medicine.