Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Abstract

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues. Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm. 6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects.In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors. Video LinkThe video component of this article can be found at https://www.jove.com/video/2925/ ProtocolThe protocols for FLIM sample preparation do not differ from those for confocal or wide-field intensity-based fluorescence microscopy. The data acquisition is followed by the main task of data analysis, i.e. extracting the fluorescence lifetimes from the raw data. Once these have been obtained, data interpretation helps to verify or falsify hypotheses. Staining cells with molecular rotors1. Prepare a stock solution (10 ml) by dissolving approximately 1 mg/ml of the dye in an appropriate solvent (e.g. methanol for BODIPY-C 12 ) 6,7 using an accurate balance and a pipette. 2. The cells (a model cancer cell line, HeLa in our case) to be stained are grown on in a multiwell plate with a coverslide underside for microscopy, in an incubator at 37 °C with a 5 % CO 2 atmosphere until ~ 80% confluent. 3. Add 10 -20 μl of the stock solution to the living cells growing in a multi-well plate (SmartSlide 50 micro-incubation system, Wafergen) in 4 ml of Opti-MEM medium (GIBCO) per well for a 6-well plate. This yields a micro-molar dye concentration in the well. 4. Return the multi-well plate to an incubator at 37 °C with a 5 % CO 2 atmosphere for 10-45 mins for staining. 5. Remove the multiwell plate from the incubator and wash the cells 3-4 times with 4 ml optically clear cell culture medium (e.g. Opti-MEM) to remove excess dye.

Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes

“…Our data show that its result is the growth of one LD, while the other one is completely with a lifetime of 1.35-1.85 ns have already been observed in FLIM images of BODIPY-C 12 -labeled human ovarian carcinoma cells ( 38 ). Due to their lifetimes, these structures have already been interpreted as LDs ( 39 ). Furthermore, another study recently published a slightly different viscosity of 34 cP inside LDs, obtained by fl uorescence correlation spectroscopy measurements ( 50 ).…”

“…The staining of the LDs with the molecular rotor BODIPY-C 12 was done according to the literature ( 39 ). In brief, a stock solution with a concentration of 1 mg/ml in methanol was prepared and 10 l of this solution was added to the living cells in petri dishes containing 2 ml of medium.…”

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements

“…The modified proteins were then incorporated into cells using the BioPORTER reagent as per the manufacturer's instructions. Flicyme analysis measures the average fluorescence lifetimes of all components within each cell within a population; therefore, we attempted to use cells in a steady state for all analyses in order to maintain a constant cell microenvironment and account for the sensitivity of fluorescence lifetime to various factors such as ionic strength, pH, and temperature [25,26]. Eventually, DyLight 680 (bioprobe for caspase-9) and Alexa Fluor 750 (bioprobe for caspase-3) were selected as acceptor molecules.…”

Recent advances in twisted intramolecular charge transfer (TICT) fluorescence and related phenomena in materials chemistry

“…Representative molecular rotors (Fig. 3) are 9-(2-carboxy-2-cyanovinyl)julolidine (6) and its analogues, 24,25 whose versatilities have been demonstrated not only in molecular biology, such as in peptide-protein interaction studies, 26 imaging intracellular microviscosity, 27 and amyloid deposit research, 28 but also in polymer science, 29 contact mechanics, 30 and fluid dynamics. 31 Thioflavin-T (7) is a well-known TICT-based molecular rotor used in in vitro cell biological and biomedical assays, in particular in drug discovery assays and mechanistical studies with respect to amyloid-related neurodegenerative diseases.…”