Fluorescence based imaging has evolved into a discipline in its own right with numerous applications in biology, clinical medicine and cancer detection. Many fundamental processes of a living cell take place at the cell surface plasma membrane, including secretion of hormones and neurotransmitters, uptake of nutrients, generation and transmission of chemical and electrical signals. Vesicular exocytosis is an important mechanism by which signaling molecules, such as hormones and neurotransmitters cross the surface membrane. The process of exocytosis-endocytosis is also involved in the recycling of membrane proteins between the plasma membrane and intracellular compartments. The ability to selectively visualize such processes and the sub-membrane structures in living cells would be tremendously helpful in improving our understanding of the fundamental principles of cell physiology and pathology. However, today no imaging method, with some exception for TIRF, is capable of directly visualizing in live cells the exocytotic events that involve transport and secretion of vesicular cargo across the plasma membrane. The size of the cell membrane is only ~10 nm and the diameter of many secretory vesicles is often well below 50 nm, thus all imaging modalities lack sufficient resolution. The long-term goal of this R21 application is to develop and validate a novel approach for monitoring membrane processes by utilizing enormous fluorescence signal enhancement resulting from near field interactions of fluorophores with surface deposited metallic nanostructures. This application builds on our recent discovery that self assembled colloidal structures (SACS) produce up to 1000- fold fluorescence signal enhancement for closely positioned fluorophores. Our immediate goal is to apply this nanophotonic phenomenon in combination with TIRF microscopy for monitoring exocytotic process with unprecedented sensitivity. This will allow close following of vesicle recruitment to the plasma membrane and kinetics of cargo release even for the smallest vesicles that typically constitute the largest portion of the secretory vesicular pool. The biological problem we want to address is the exocytotic ATP release from non-excitable, cancerous lung epithelial cells. TIRF microscopy will visualize vesicles loaded with fluorescently-tagged ATP by evanescence field excitation in distances up to 200 nm. Following the exocytotic release into the extracellular space the labelled cargo will be positioned within the enhancement field of the metallic nanostructure, producing a huge, easy to detect signal that disappears due to free diffusion of the fluorophore into the extracellular space.

Public Health Relevance

ATP release is suggested to take place at the leading edge of a migrating cell, contributing to a coordination of many processes that are involved in a directional cell movement. Proposed nanophotonic approach will allow studying these complex processes with unprescedented sensitivity and help to better understand regulation of tumour metastasis (tumour spread) and invasiveness.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Exploratory/Developmental Grants (R21)
Project #
Application #
Study Section
Medical Imaging Study Section (MEDI)
Program Officer
Knowlton, John R
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of North Texas
Other Domestic Higher Education
Fort Worth
United States
Zip Code
Shumilov, Dmytro; Popov, Alexander; Fudala, Rafal et al. (2014) Real-time imaging of exocytotic mucin release and swelling in Calu-3 cells using acridine orange. Methods 66:312-24
Seo, Jaetae; Fudala, Rafal; Kim, Wan-Joong et al. (2012) Hybrid optical materials of plasmon-coupled CdSe/ZnS coreshells for photonic applications. Opt Mater Express 2:1026-1039
Maliwal, Badri P; Raut, Sangram; Fudala, Rafal et al. (2012) Extending Förster resonance energy transfer measurements beyond 100 Å using common organic fluorophores: enhanced transfer in the presence of multiple acceptors. J Biomed Opt 17:011006
Fudala, Rafal; Mummert, Mark E; Gryczynski, Zygmunt et al. (2012) Lifetime-based sensing of the hyaluronidase using fluorescein labeled hyaluronic acid. J Photochem Photobiol B 106:69-73
Ding, Yanfeng; Winters, Ali; Ding, Min et al. (2011) Reactive oxygen species-mediated TRPC6 protein activation in vascular myocytes, a mechanism for vasoconstrictor-regulated vascular tone. J Biol Chem 286:31799-809
Fudala, Rafal; Mummert, Mark E; Gryczynski, Zygmunt et al. (2011) Fluorescence detection of hyaluronidase. J Photochem Photobiol B 104:473-7
Luchowski, Rafal; Calander, Nils; Shtoyko, Tanya et al. (2010) Plasmonic platforms of self-assembled silver nanostructures in application to fluorescence. J Nanophotonics 4:
Luchowski, Rafal; Szabelski, Mariusz; Sarkar, Pabak et al. (2010) Fluorescence instrument response standards in two-photon time-resolved spectroscopy. Appl Spectrosc 64:918-22
Luchowski, Rafal; Sabnis, Sushant; Szabelski, Mariusz et al. (2010) Self-quenching of uranin: Instrument response function for color sensitive photo-detectors. J Lumin 130:2446-2451