**1. Introduction**

Cells secrete proteins into the extracellular environment to achieve important physiological processes, such as transportation of nutrients, digestion of food, regulation of metabolic processes, etc [1–5]. Cell secretion can be classified into the constitutive and regulated secretory pathway [6–8]. The constitutive secretory pathway is associated with transportation of secretory vesicles to the cell surface and their release independently of stimulus [9–11]. The regulated secretory pathway produces secretory granules, stores them in the cytoplasm, and secretes them into the extracellular environment only upon receiving stimuli [12]. In human, more than 15% of the genome encoded proteins are associated with the secretion process [13]. These released proteins include neurotransmitters, protein hormones, growth factors, cytokines, enzymes, antibodies, etc [14]. They play a vital role in intercellular communication [15], which further regulates cell functions in immunology [16–18], neurobiology [19–21], endocrinology [22–24], etc. For instance, as the immune cells are stimulated, they undergo dynamic alternation and secrete various types of cytokines in a heterogenous manner. These secreted cytokines regulate the maturation and growth of immune cells, and activate the immune effector and memory immune responses.

The heterogeneity of cellular protein secretion has been found in various human diseases, including malignant proliferation of cells, aberrant immune function, bone marrow failure, etc [25–27]. This heterogeneity is present between distinct cell populations such as normal and pathological cells, as well as the same phenotype of cells [28–30]. This secretome heterogeneity creates diverse microenvironment for cells. The proliferation and metastasis of tumor cells can be prohibited or promoted upon the discrepancy of microenvironment, in spite of within the same tumor [31]. Meanwhile, the secretome change is frequently associated with atypical cellular phenotypes [32], which are indicative of diseases such as cancers, thus can be used as significant markers for tumorigenic process at single cell level. Therefore, characterization of protein secretion from single cell play a vital role in not only understanding the intercellular communication in immune effector, carcinogenesis and metastasis, but also developing and improving the current diagnosis and therapy of relative diseases [33–35]. In spite of abundant highly sensitive methods that have been developed for the detection of secreted proteins, majority of them have insufficient resolution in space and time, thus are not capable of providing deep insight into the behavior of protein secretion from single cells. Ideally, a detection method is supposed to be equipped with a high spatial resolution that enables differentiation of single cells from each other, and visualization of the spatial distribution of secreted proteins among multiple single cells, as well as a high temporal resolution that can recognize the dynamic alteration of secretion in a quantitative manner. The realtime imaging techniques feature rapid acquisition of analyte information on a 2D plane, and high spatial and temporal resolution, thus attract considerable attention in recent years, for its high potential to realize the deep exploration of single cellular protein secretion. Here, we review the involved works towards real-time imaging of single cell's protein secretion, including the detection of single or multiplex protein secretion, from group cells or single cells, in a real-time or near real-time manner, analyze their advantages and limitations, and discuss their major challenges.
