Multiplexed Fluorescence Lifetime Microscopy of Live Cells Using Fluorogen-Activating Proteins
Alexey M. Bogdanov1; Yulia A. Bogdanova2, IlyaD. Solovyev3, Nadezhda S. Baleeva2,4, Ivan N. Myasnyanko2,4, Anastasia A. Gorshkova2, Dmitriy A. Gorbachev2, Aidar R. Gilvanov2, Sergey A. Goncharuk2, Marina V. Goncharuk2, Konstantin S. Mineev2,6, Alexander S. Arseniev2, , Alexander P. Savitsky3, Mikhail S. Baranov2,4,5; 1Department of Photonics, İzmir Institute of Technology, İzmir, Turkey; 2Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia; 3A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia; 4Pirogov Russian National Research Medical University, Moscow, Russia; 5Department of Biology, Lomonosov Moscow State University, Moscow, Russia; 6Present address: Goethe University Frankfurt, Frankfurt am Main, Germany
Abstract
In this lecture, we provide details on the recently proposed fluorescence-lifetime imaging microscopy (FLIM) multiplexing systems based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform is overviewed within the broad context of biological microscopy techniques, with emphasis on image contrast enhancement, advancements and shortcomings of diverse fluorescence labeling approaches. After a brief introduction to label-free and label-based imaging modalities, we explain the strengths of fluorescence labeling in cellulo and discuss the principles of multiplexed visualization. We familiarize the audience with state-of-the-art time-resolved fluorescence techniques, paying special attention to those based on genetically encoded labeling platforms, including chemogenetic fluorophores. Finally, we discuss the platforms employing FAST (fluorescence-activating and absorption-shifting tag) protein variants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair.
Speaker
Alexey Bogdanov
Izmir Institute of Technology
Turkey
Discussion
Lomova Maria
What are the limitations of the research samples using the method you described?
Alexey Bogdanov
The major drawback of this technique is the necessity of externally adding fluorogens. These dyes can potentially disturb cellular physiology, and their ability to reach deep-tissue regions during in vivo and ex vivo experimentation is quite limited.
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