![]() This is not possible using conventional flow cytometry as similar emission spectra would be detected in the same channel. A distinct advantage compared with conventional multicolor flow cytometry is that, as long as fluorophores have distinct signatures across the spectrum, fluorophores with similar peak emission, like APC and Alexa Fluor 647, can be used in the same panel (Ferrer-Font et al. This happens through a process called spectral unmixing and involves complex mathematical models that employ an unmixing algorithm. In a multiparameter experiment, once you have collected the full spectrum data, individual fluorophores need to have their profiles isolated. How Do You Identify Individual Fluorophores? Spectral detectors ( bottom) use gratings or prisms to separate light and a collimating lens to parallelize and direct light linearly across an array of detectors. Conventional detectors ( top) use dichroic mirrors, bandpass, and long pass filters to separate colors of light for individual detection. Simplified comparison of conventional and spectral flow cytometer instruments for a 2-laser system (Adapted from Nolan and Condello 2013). This means that while conventional flow cytometry effectively detects signals from specific fluorophores over defined wavelengths, spectral flow cytometry instead collects the entire spectral profile (also called a spectral signature) of fluorophores from multiple lasers.įig. Spectral flow cytometry differs from conventional flow cytometry as it uses prisms to capture all the emitted light from laser excited fluorophores across a set of detectors, or an array of channels, rather than detecting emitted photons that are collected into individual detectors (Figure 1). Spectral flow cytometry is a rapidly growing area of flow cytometry and, in this blog, we look at the basic principles and the advantages over conventional flow cytometry. 2004), with a patent issued for the technology to Purdue University in 2007 (Robinson 2019). The application of spectral flow cytometry was initially presented by the Purdue group at the 2004 International Society for Analytical Cytology (ISAC) meeting (Robinson et al. ![]()
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