Cell Imaging by Laser-Induced Native Fluorescence Microscopy

Abstract

The high degree of heterogeneity of the nervous and endocrine systems makes it extremely important for real-time monitoring of dynamic chemical changes at the single-cell level to gain a better understanding of the interaction of cells with their environment. Secretion mediated by exocytosis is one of the fundamental phenomena whose mechanism mimics the release of neurotransmitters at synaptic sites. Although the regulation of the secretory pathway has been studied extensively, its molecular mechanism is still not clear. It is important to develop methods that can follow real-time secretory processes with both high temporal and high spatial resolution. The native fluorescence of some proteins and neurotransmitters excited by a deep-UV laser has been shown to be a powerful probe for single-cell analysis. The advantages of direct native fluorescence detection include: (1) no chemical derivatization with fluorescent dyes is needed so no contamination or additional background will be introduced; (2) uncertainties about the efficiencies of the derivatization reaction are eliminated to ensure fast and quantitative response, without influences from slow reaction kinetics or incomplete equilibrium; and (3) the biological integrity of the cells will not be unnecessarily disturbed by having additional reagents or from exposure to artificial environments. We report the coupling of laser-induced native fluorescence detection with capillary electrophoresis (CE) to quantitatively monitor the secretion of insulin, serotonin and catecholamines from single cells. The uptake of serotonin by single living astrocytes was also recorded by native fluorescence imaging microscopy. The catecholamine (mainly epinephrine and norepinephrine) secreting adrenal chromaffin cells have been used as “model nerve terminals” to elucidate the molecular mechanism of neurotransmitter secretion at the nerve terminal. The in vitro dynamics of catecholamine release from bovine adrenal chromaffin cells was monitored with both high spatial and high temporal resolution.