Photophysical Insights and Polarity Dynamics of 4-Diethylamino Salicylaldehyde-Based Fluorescent Molecules and Their Application as Mitochondrial and Endoplasmic Reticulum Tracker Agents

4-Diethylamino salicylaldehyde group offers tunable structural and photophysical properties and chemical versatility, making it a valuable scaffold in fluorescence-based imaging studies. The electronic properties of the molecule can be altered by various reactions, including Schiff base formation, −C=C condensation reaction, coumarin formation, etc. Thus, we explored the structural properties and polarity parameters of two core molecules, P1 and P3, based on the 4-diethylamino salicylaldehyde core group and its further derivatives, P2 and P4. The fluorescence behavior of P1 and P3 was well explained by correlating their photoelectron transfer (PET) and intramolecular charge transfer (ICT) mechanisms, respectively. Fluorescence behavior of P1 and P3 was further studied by increasing the solvent’s viscosity in the glycerol–ethanol mixture. Furthermore, P2 and P3 were utilized as mitochondria (Mito) and endoplasmic reticulum (ER) trackers as they possessed a cationic site in the structure that can interact with the negative charge potential in these organelles. Thus, we developed efficient, noncytotoxic, affordable, and easily synthesized fluorescent molecules for Mito and ER tracking, enabling the study of cellular processes, disease diagnosis, and drug evaluation at the organelle level.