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Wade and colleagues made one of the first attempts to extract full emission spectra during flow cytometry analysis in 1979. It will allow better separation of negative, dim, and positive populations using multicolor labeling. Besides, significant progress in the decomposition of complex fluorescent spectra was introduced by Rosetti and co-authors, which could improve spectral unmixing and detection of autofluorescence.

Recent technical advances in multicolor cytometry were focused on detecting and analyzing cellular subpopulations with complex immunophenotypes participating in the immune response to diseases and/or vaccine response. Both imaging flow cytometry and spectral FCM allow sophisticated offline analysis of the specimens.

Spectral FCM, which is based on spectroscopy, made it possible to record the full spectrum of every single cell during measurements and now operates at a rate similar to conventional flow cytometry. However, its throughput is significantly less than conventional flow cytometry. Imaging flow cytometry, a hybrid technology, which combines the principles of flow cytometry and microscopy, allows obtaining an image of each cell and thus collects galleries of images along with light scatter and fluorescent signals.

As it developed, flow cytometry in the twenty-first century diverges into the following directions: (1) Conventional flow cytometry and fluorescent activated cell sorting (FACS) (2) Imaging flow cytometry (3) Spectral flow cytometry (spectral FCM).Ĭonventional cytometry allows studying the size, granularity, and several fluorescent signals of individual cells or particles at the rate of 1000 events per second. Flow cytometry began its development in the middle of the twentieth century and has established itself as one of the major functional methods widely used by scientists and clinicians.

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