Quantitative scanning-free confocal fluorescence microscopy for the characterization of fast dynamic processes in live cells
- Abstract number
- 278
- DOI
- 10.22443/rms.mmc2021.278
- Corresponding Email
- [email protected]
- Session
- Stream 6 (Frontiers): Quantifying Dynamic Movement in Living Cells
- Authors
- Dr Sho Oasa (1), Dr Aleksandar J. Krmpot (3), Dr Stanko N. Nikolic (3), Prof. Andrew H. A. Clayton (4), Prof. Lars Terenius (1), Prof. Rudolf Rigler (2), Assoc. Prof. Vladana Vukojevic (1)
- Affiliations
-
1. Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM), Karolinska Institutet
2. Department of Medical Biochemistry and Biophysics (MBB), Karolinska Institute
3. Institute of Physics Belgrade, University of Belgrade
4. Optical Sciences Centre, Department of Physics and Astronomy, School of Science, Swinburne University of Technology
- Keywords
Scanning-free Confocal Fluorescence Microscopy, Functional Fluorescence Microscopy Imaging (fFMI), Fluorescence Correlation Spectroscopy (FCS), Fluorescence Lifetime Imaging Microscopy (FLIM), Biomolecular Interactions, Dissociation Constants, Transcription Factors, G Protein-Coupled Receptors (GPCRs), Dissociation constant
- Abstract text
To understand how complex biological functions emerge through random motion of molecules and their chemical reactions, the concentration and mobility of biological molecules need to be quantitatively characterized in live cells. To this aim, Fluorescence Correlation Spectroscopy (FCS) is uniquely available. However, conventional FCS is hampered by limited overview, as it allows single-point measurements only. It also does not give information about physiological consequences of the characterized molecular interactions. To overcome these limitations, we have recently developed massively parallel FCS (mpFCS) [1]. We have now integrated mpFCS with Fluorescence Lifetime Imaging Microscopy (mpFCS/FLIM), thus obtaining a new functional Fluorescence Microscopy Imaging (fFMI) modality. Our instrument is a quantitative scanning-free confocal fluorescence microscope that enables us to characterize the cellular dynamics of molecules with a temporal resolution of 10 µs/frame, single-molecule sensitivity and map fluorescence lifetimes from 1 ns – 10 ns. In my talk, I will present this new methodology and its utility for live cell molecular studies and single-cell pharmacology.
- References
1. Krmpot AJ, Nikolić SN, Oasa S, Papadopoulos DK, Vitali M, Oura M, Mikuni S, Thyberg P, Tisa S, Kinjo M, Nilsson L, Terenius L, Rigler R, Vukojević V. Functional Fluorescence Microscopy Imaging (fFMI). Quantitative Scanning-Free Confocal Fluorescence Microscopy for the Characterization of Fast Dynamic Processes in Live Cells. Anal. Chem. 2019 91(17):11129-11137.