and T.E.H. However, BRET is generally not compatible with high-resolution microscopy-based imaging due to low photon yield. Numerous efforts have been made to develop improved luciferase CD140b enzymes, better suited to bioluminescence imaging, such as Nano-luciferase (Nluc) which produces an intense and sustained luminescence with high transmission stability and luminescence efficiency as shown with the Calflux calcium-reporting biosensor [13,34]. Nluc allows luminescence quantification from small numbers of molecules, and is bright enough for single cell BRET imaging applications . For instance, engineering Nluc into a biosensor that reports ERK1/2 activity has shown to improve the sensitivity as well as the temporal resolution of the BRET signals acquired . Table 1 Advantages and disadvantages of RET techniques- bioluminescence versus fluorescence resonance energy transfer-based sensors. (This has been extensively examined in Kauk et al. ). Studies Genetically-encoded biosensors can reveal fine spatial and temporal details of L-Tryptophan cellular signaling processes and have provided a rich understanding of the pluridimensionality of these processes in model systems. However, the power of such data for predicting therapeutic drug action depends entirely on how well the chosen model displays the physiological and pathological fact of the disease in question. The application of optical biosensors together with single cell methods can reveal the granularity of individual cell responses and suggests a link between specific cell says and receptor-mediated signaling over a large populace. 3.1. Single Cell Sequencing The expression profile of GPCRs within a specific tissue type is usually variable and often dynamic in nature. Recent examinations of GPCR expression in main vascular smooth muscle mass cells, vascular endothelial cells, T cells and myeloid cells have shown that within a single cell type, GPCR L-Tryptophan expression profiles are variable and may be altered during development, tissue engineering or in disease says [47,48,49,50,51]. A better understanding of this aspect of cellular context can lead to more efficient drug targeting of cells expressing a disease phenotype. Beyond differential expression of receptors themselves, changes L-Tryptophan in the stoichiometry L-Tryptophan or activity of signaling partner proteins such as G proteins can also impact signaling responses. For example, D2 dopamine receptors (D2R) found on medium-spiny neurons of the dorsal and ventral striatum display different sensitivities to dopamine agonists due to differential expression of the G subunits Gi and Go . Similarly, in -opioid receptor-expressing neurons from dorsal root ganglia, two unique signaling populations can be delineated based on their responses to morphine, and the difference between signaling groups was dependent on protein kinase C activity , suggesting a role for downstream effectors in determining this response. These studies demonstrate that classically defined cell taxonomies based on morphological or incomplete sets of genetic markers may not capture the potential granularity in the signaling behavior of cells which are considered to be a single cell type. To date, cell type characterization has been dependent on specific cellular behaviors or the expression of relevant molecular markers. Based on the latter, population-based assays have often been performed examining responses in cell populations defined by a set of specific markers. While limitations of this cell type identification criteria were known, the full impact of transcriptomic and functional heterogeneity in cell populations has only recently begun to be appreciated. Through initiatives such as the Allen Brain Atlas, heterogeneous gene expression patterns in the mouse brain have L-Tryptophan started to be unraveled ..