In vitro analysis of intramolecular signaling events in PDLSCs using confocal and TIRF microscopy

Abstract

The advances in microscopy techniques enable the detection of intracellular molecular processes to be visualized and analyzed for periodontal ligament stem cells (PDLSCs). Confocal laser scanning microscopy (CLSM) and total internal reflection fluorescence microscopy (TIRFM) are two well-studied microscopy techniques that allow an increase in the resolution and contrast of the micrographs and the capability to pinpoint events at the plasma membrane, respectively. Confocal microscopy achieves its increased resolution and contrast through a spatial pinhole that hits the plane of the image. TIRF microscopy uses the principle of incident angles and the refractive index of the substances to study the events occurring at 100–200 nm range of the cover glass with minimal background interference. Here we describe two methods for intramolecular signaling visualization upon stimulation with a ligand in normal growth conditions and mineralization-induced conditions in periodontal ligament stem cells (PDLSCs). These methods are important for visualizing the signaling events within PDLSCs at a molecular level and thereby understand the mechanisms by which these cells could be manipulated for tissue engineering and regeneration.