Recent studies show that IFC allows to localize and quantitate molecules (peptides, proteins, protein complexes, nucleic acids (DNA, RNA, miRNA), glycolipids, molecular assemblages, etc.) not only in the single cells, but also in multicellular clusters and small aggregates and to map their spatial distribution to the cellular compartments. Single cell measurements allow revealing information hidden in population averages.

Using fluorescent molecules as markers allows IFC to perform analysis of host-pathogen interaction, quantification of intracellular parasites and has a potential of providing high-throughput analysis of host-protein interactions and assessment of drugs for parasitic and microbial infections [16, 17].

IFC is extremely helpful in the cases when live cells could be associated with dead cells and apoptotic bodies [3], adherent platelets/platelet fragments [4], or extracellular vesicles (EVs). In these situations use of conventional flow cytometric techniques and excluding events only by FSC/SSC parameters may result in the interpretation of negatively stained cells as positive ones. This was shown for apoptotic markers [18] and/or the CD41a⁺ expression [19]. CD41a is a glycoprotein (Ca⁺⁺-dependent heterodimer) that serves as a receptor for adhesion proteins on the surface of platelets and mediate cell-to-substrate and cell-to-cell adhesion. Many cancer cells floating in the blood vessels are temporarily coated with platelets, possible to escape immune surveillance [19, 20] and their analysis by IFC is the most promising approach.

IFC is also very helpful in distinguishing false-positive events in cell death evaluation [21] and during analysis of activated cells. The cellular suspensions always have level of spontaneous apoptosis >0 % and constantly produce extracellular vesicles (EVs) in response to apoptotic and activation stimuli. The EVs are lipid vesicles produced by all cells, and can carry nucleic acids and a number of proteins originated from cell-progenitors. Therefore EVs can be positively stained with DNA and lipid dyes, and/or fluorochrome-conjugated antibodies and attach to negatively stained cells. Annexin V⁺ staining is a marker of significant subset of EVs (microparticles, size range 100–1000 nm). Only IFC allows verification and exclusion of a subset of cells with EVs attached to their surface and obtaining proper statistics on the cell populations.