Fluorescence Lifetime Imaging Microscopy

In the present work, we report the imaging of Au nanostars nanoparticles (AuNSt) and their multifunctional applications in biomedical research and theranostics applications. Their optical and spectroscopic properties are considered for the multimodal imaging purpose. The AuNSt are prepared by the seed-meditated method and characterized for use as an agent for bio-imaging. To demonstrate imaging with AuNSt, penetration and localization in different biological models such as cancer cell culture (A549 lung carcinoma cell), 3D tissue model (multicellular tumor spheroid on the base of human oral squamous carcinoma cell, SAS) and murine skin tissue are studied. AuNSt were visualized using fluorescence lifetime imaging (FLIM) at two-photon excitation with a pulse duration 140 fs, repetition rate 80 MHz and 780 nm wavelength femtosecond laser. Strong emission of AuNSt at two-photon excitation in the near infrared range and fluorescence lifetime less than 0.5 ns were observed. It allows using AuNSt as a fluorescent marker at two-photon fluorescence microscopy and lifetime imaging (FLIM). It was shown that AuNSt can be observed inside a thick sample (tissue and its model). This is the first demonstration using AuNSt as an imaging agent for FLIM at two-photon excitation in biosystems. Increased scattering of near-infrared light upon excitation of AuNSt surface plasmon oscillation was also observed and rendered using a possible contrast agent for optical coherence tomography (OCT). AuNSt detection in a biological system using FLIM is compared with OCT on the model of AuNSt penetrating into animal skin. The AuNSt application for multimodal imaging is discussed.

BACE is a transmembrane protease with beta-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for gamma-secretase, leading to release of amyloid-beta peptide (Abeta), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate.

The p38 MAPK and heat shock protein 27 (hsp27) form a signaling complex with serine/threonine kinase Akt and MAPK-activated protein kinase-2 (MK2), which plays an important role in controlling stress-induced apoptosis and reorganizing actin cytoskeleton. However, regulation of the complex is poorly understood. In this study, the interaction between p38 and hsp27 was visualized in single living L929 cells using fluorescence resonance energy transfer technology, while their association with Akt was examined by immunoprecipitation analysis. Under normal growth conditions, p38 kinase constitutively interacts with hsp27. When cells were exposed to H(2)O(2) or stimulated by arachidonic acid, this interaction was disrupted. However, inhibition of the activation of p38 and Akt by selective inhibitors or overexpression of the kinase-dead mutant of p38 diminished such effects. Furthermore, mutation of phosphorylation sites of hsp27 renders the interaction resistant to H(2)O(2) and arachidonic acid. It was interesting to find that the interaction disappeared in the cells from MK2-knock-out mice or the cells treated with lemptomycin B that blocks export of MK2 from nucleus to cytosol. However, MK2 is not required for the association of hsp27 with Akt. This study suggests that MK2 mediates the incorporation of p38 into the pre-existing complex of hsp27 with Akt. Phosphorylation of hsp27 finally breaks the signaling complex.

Metabolism is a complex phenomenon in the biological systems, falling into one of two general categories: catabolic and anabolic reactions, or the processes of breaking down and building up, respectively. Changes in cell or tissue metabolic states respond to changes in physiological demands or the onset of pathological conditions. Auto-fluorescent endogenous biomolecules associated with metabolism such as coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are key players in the production of adenosine triphosphate (ATP)—the energy currency of life. Thus, changes in the abundance of NADH and FAD can serve as noninvasive biomarkers in cells and tissues. There are other auto-fluorescent endogenous molecules, but none as thoroughly investigated as NADH and FAD by advancements in technology such as intensity and lifetime microscopy. The importance of the coenzymes NADH and FAD in metabolism was fully recognized to the scientific world by the great innovator, the late Prof. Britton Chance (1913–2010). He demonstrated cellular NADH auto-fluorescence in 1958 by fluorescence spectrophotometry 1. He developed a fiber optics based fluorimeter to monitor by NADH, the mitochondrial metabolic state of awake rat brains during 1962 2. He has published over 1,500 research articles. Prof. Chance's passion was in translational research and mentoring diverse student populations—regardless of their gender, ethnicity, nationality, religion, or social status—to influence research communities all over the world. Prof. Chance students regarded him as a role model, great mentor, and dear friend. Prof. Chance has tirelessly promoted scientific cooperation and academic exchanges between East and West 3. The ability to make these visible the endogenous biomarkers introduced by Prof. Chance led to their characterization and pathways. During glycolysis, two molecules of ATP are produced in the cytoplasm. Cytoplasmic NADH is transported to mitochondria by malate–aspartate shuttle 4 where, NADH is oxidized to NAD+ producing malate; malate is transported to mitochondrial matrix, mitochondrial NAD+ is reduced to NADH which is in turn is oxidized to NAD+ by the electron transport chain (ETC) in the respiratory chain in the mitochondrial matrix 4, 5. Mitochondrial NADH is produced by citric acid cycle or Kreb's cycle based on several substrates. The energy stored in mitochondrial NADH is transferred to an electrochemical proton gradient that is essential to drive phosphorylation of ADP to ATP by F0F1 ATP synthase, to produce approximately 30 units of ATP, the energy currency of the cell (see Fig. 1 reproduced from the review by Schaefer et al. [in this issue, 34]). The coenzymes FMN/FAD are derived from riboflavin (vitamin B 2) and are covalently bound to flavoproteins in enzymatic reactions of the Kreb's cycle and ETC which takes place in the mitochondria 4. The ETC complex II of succinate dehydrogenase contains bound FAD cofactor which is reduced to FADH2. FADH2 is then oxidized to FAD by Coenzyme Q 4, 5. Only the oxidized form of the flavins, FpOx (FAD or FMN) are fluorescent whereas, the reduced forms Fp (FADH2 or FMNH2), are nonfluorescent. On the other hand, NAD(P)H is fluorescent whereas, the oxidized form NAD(P)+ is nonfluorescent 5, 6. Three out of the 20 amino acids, Tyrosine (Try or Y), Phenylalanine (Phe or F), and Tryptophan (Trp or W), all aromatic, are autofluorescent absorbing and emitting in the UV region. Tryptophan (Trp), an essential amino acid is of particular interest in the biomedical sciences, their fluorescence intensity and lifetime providing information on protein abundance and is dysregulated in cancer 7, 8. Trp is moderately fluorescent with a quantum yield of 0.1–0.5 and has the highest absorption coefficient of the 3 amino acids, and therefore the highest brightness and the longest emission wavelength of 350 nm 8. Alterations in the tryptophan metabolism are suggested to be a common pathological pathway for clinically distinct neurodegenerative disorders, like Parkinson's, Huntington's, and Alzheimer's diseases 9. Modern technologies such as fluorescence lifetime imaging (FLIM) produce endogenous fluorophore data as readouts of mitochondrial function in their natural environment for investigators in the biological and clinical sciences. The unique characteristics of lifetime are that it is sensitive to environmental changes and insensitive to change in fluorophore concentration or excitation intensity. Lifetime helps investigators to fingerprint the molecular functions under various conditions 10. Lifetime techniques have been used for measuring NADH decays since 1967 by spectrofluorometry 11. FLIM provides both spatial and temporal information of molecules and discriminates the bound from free NADH (ref) and NADH from NAD(P)H 12. In this special Cytometry A issue on Metabolism, 10 well-established authors used multiple imaging techniques to explore metabolism in various directions, by: different cell types in 2D and 3D, single cell analyses and their intra- and inter-cell heterogeneities, response to treatment. While imaging the auto-fluorescent NADH and FAD molecules have come a long way to provide insights into the metabolic states of cells and tissue, Schaefer et al. (in this issue, 34) in their very thorough review discuss the “confounding factors” and potential pitfalls—and how to mitigate them. Lukina et al. (in this issue, 47) and Cao et al. (in this issue, 100) discuss additional correlative biomarkers to NADH in second harmonic generation (SHG) of collagen and the putative FRET interaction of Tryptophan (Trp) and NADH, respectively, both in tumors. Lukina et al. (in this issue, 47) investigated the cellular metabolism collagen in colorectal cancer model. Their results demonstrates that the changes in cellular metabolism and fibrotic stroma organization precede morphological alterations and tumor size reduction, and that indicates that NAD(P)H and collagen can be considered as intrinsic indicators of the response to colorectal cancer treatment. Cao et al. (in this issue, 110) presents data in HeLa cervical cancer cells upon treatment with doxorubicin to show the correlations of Trp-NADH energy transfer (E%) of putative Trp residues on enzymes interacting with NADH during NADH to NAD+ transition to a novel, entirely FLIM-based redox ratio – FLIRR 13. In addition, the NADPH/NADH ratio as per Blacker 12 is established suggesting the changing balance between catabolism and anabolism/ROS defense. Cong et al. (in this issue, 80) applied 2-photon FLIM to characterize the critical factors such as physical and chemical microenvironmental signaling in in vitro 3-dimensional tumor models. Ramanujan (in this issue, 101) used a flow cytometry assay to monitor NAD(p)H oxidation kinetics and metabolic status in human breast cancer cells. He also investigated tumor cell heterogeneity in primary tumor epithelial cells isolated from preclinical animal models. Ma et al. (in this issue, 93) sorted out the leukemia cell lines based on their metabolic state differences between proliferation and quiescent cells using the droplet microfluidic technology and phasor FLIM. This approach may be useful to upstream phenotypic platform to correlate with genomic statistics. Chorvatova et al. (in this issue, 13) provided a detailed analysis of endogenous flavins fluorescence photobleaching arising in living cardiac cells during spectrally resolved confocal imaging. This investigation led to significant modifications of the cell responsiveness to metabolic modulators and its contractility, suggesting functional metabolic alterations in the recorded cells. FLIM based AFI imaging is a sensitive way to monitor the cellular metabolism but there are many issues involved in the complex biological system and that may influence the variability in the lifetime values. Chacko and Eliceiri (in this issue, 56) described in this article that it is important to consider the cellular microenvironment such as confluency, pH, inter-/intra-cellular heterogeneity, and choice of cell line for accurate quantitative FLIM based AFI measurement of cellular metabolism. Altarkistany et al. (in this issue, 70) monitored the shift in the fluorescence decay of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis using cytometry based FLIM acquisition with phasor analysis. They have demonstrated free to bound ratios as the ratio of “short-to-long” (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. This special issue on metabolism highlights the challenges involved in the investigation using biomarkers but also provides great opportunities to advance research by using the advanced technologies such as FLIM and data analysis at single cells for tissue/in vivo metabolism mapping using biomarkers such as FLIRR, Tryptophan and SHG for changes in collagen. We would like to thank Dr. Attila Tarnok, Editor-in-Chief and Steven Perez, Associate Managing Editor for their valuable guidance and help during this administration of this special issue.

Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation is emerging as a reliable quantitative tool for measuring fluorescence resonance energy transfer (FRET) in living cells. By virtue of being free from spectroscopic artifacts encountered in conventional FRET detection methods, multiphoton FLIM methods offer the advantages of high spatial and temporal resolution, faster data acquisition and data analysis. We compare the FRET results obtained by two different methods namely (i) multiphoton excitation lifetime-based FRET and (ii) single photon excitation intensity-based acceptor photobleaching FRET. Using the same biological samples, we apply these two different methods in understanding the growth hormone receptor dimerization kinetics at the cell surface of human embryonic kidney cells. We conclude that the multiphoton FLIM using the streak-camera approach provides the best ability to monitor FRET in dynamic situations where high temporal and spatial resolution are required with minimal photodamage/phototoxicity.

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.

Utilizing fluorescent life time imaging microscopy technology for identify carriers of BRCA2 mutation

Fluorescence lifetime imaging microscopy (FLIM) has been widely used in the field of biological research because of its high specificity, sensitivity, and quantitative ability in the sensing cellular microenvironment. The most commonly used FLIM technology is based on time-correlated single photon counting (TCSPC). Although the TCSPC method has the highest temporal resolution, the data acquisition time is usually long, and the imaging speed is slow. In this work, we proposed a fast FLIM technology for fluorescence lifetime tracking and imaging of single moving particles, named single particle tracking FLIM (SPT-FLIM). We used feedback-controlled addressing scanning and Mosaic FLIM mode imaging to reduce the number of scanned pixels and the data readout time, respectively. Moreover, we developed a compressed sensing analysis algorithm based on alternating descent conditional gradient (ADCG) for low-photon-count data. We applied the ADCG-FLIM algorithm on both simulated and experimental datasets to evaluate its performance. The results showed that ADCG-FLIM could achieve reliable lifetime estimation with high accuracy and precision in the case of a photon count less than 100. By reducing the photon count requirement for each pixel from, typically, 1000 to 100, the acquisition time for a single frame lifetime image could be significantly shortened, and the imaging speed could be improved to a great extent. On this basis, we obtained lifetime trajectories of moving fluorescent beads using the SPT-FLIM technique. Overall, our work offers a powerful tool for fluorescence lifetime tracking and imaging of single moving particles, which will promote the application of TCSPC-FLIM in biological research.

Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5–14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54–1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.

Protein localization in living cells and tissues using FRET and FLIM

The biological interpretation of the genome starts from transcription, and many different signaling pathways are integrated at this level. Transcription factors play a central role in the transcription process, because they select the down-stream genes and determine their spatial and temporal expression. In higher eudicot species around 2000 specific transcription factors are present, which can be classified into families based on conserved common domains. The MADS-box transcription factor family is an important family of transcription regulators in plants and genetic studies revealed that members of this family are involved in various developmental processes, like floral induction, floral organ formation and fruit development. In contrast to this wealth of information concerning MADS-box gene functions, the molecular mode of action of the encoded proteins is far from completely understood. Biochemical and yeast η-hybrid experiments performed in the past showed that MADS-box proteins are able to interact mutually, and based on these findings a hypothetical quaternary model has been proposed as molecular working mechanism. According to this model two MADS-box protein dimers assemble into a higher order complex, which binds DNA and regulates target gene expression. Although, this molecular mechanism sounds plausible, it still lacks evidence from in vivo studies. In this study we investigated physical interactions among members of the Petunia hybrida and Arabidopsis thaliana MADS-box transcription factor families in living plant cells. For this purpose, sophisticated micro-spectroscopy techniques have been implemented and in addition, some novel fluorescent-protein-based tools were developed. The first chapter gives an introduction about the dynamic transcriptional process and describes our current knowledge about transcriptional regulation in eukaryotes. The central question of this chapter is how transcription factors are able to find their specific binding sites (ci's-elements) within the huge genome. The various mechanisms, such as "looping" and "sliding", that have been proposed are discussed, as well as the relevance of direct interactions between transcription factors for the control of gene expression. In a first attempt to detect protein interactions in living cells, we transiently expressed combinations of petunia MADS-box transcription factors labeled with different color variants of the Green Fluorescent Protein (GFP) in leaf protoplasts (Chapter 2). Subsequently, the transfected protoplasts were analyzed by means of FRET-FLIM (Fluorescence Resonance Energy Transfer - Fluorescence Lifetime Imaging) to identify specific dimerization. In addition, we have obtained indirect evidence for higher-order complex formation of the petunia MADS-box proteins FLORAL BINDING PROTEIN2 (FBP2), FBP11, and FBP24 in living cells. Similar kind of analyses for Arabidopsis MADS-box proteins involved in petal and stamen development revealed clear differences in interaction affinities in vivo and furthermore, many homodimers were identified that could not be detected by yeast-based systems in the past (Chapter 3). This result demonstrated the robustness of the FRET-FLIM approach. Based on our observations, we hypothesize that 'partner selectivity' plays an important role in complex formation at particular developmental stages. To study differences in interaction affinity and selectivity and the consequences for complex formation in more detail, a novel method was developed (Chapter 4). The technique, designated "Competition-FRET", allows the verification of competition effects between proteins, and furthermore, it may provide information about the formation of higher-order complexes between different proteins under study. The developed method was implemented to investigate in depth the preference for homo- or heterodimer interactions of the Arabidopsis MADS-box proteins AGAMOUS (AG) and SEPALLATA3 (SEP3). The detection of interactions in living cells by FRET as it has been done in the studies described above demands a sophisticated microscopy set-up, and therefore, we decided to test and implement an alternative and theoretically simple technique (Chapter 5). This method for the in vivo detection of protein-protein interaction is called BiFC (Bimolecular Fluorescence Complementation), or "Split-YFP". In this system, a fluorescent molecule is split into two inactive domains and these two non-fluorescent parts are fused to the proteins under study. Only upon interaction of the two protein partners the two non-fluorescent parts of the fluorescent molecule are brought into close proximity, which enables the recovery of fluorescence. We used the EYFP (Enhanced Yellow Fluorescence Protein) molecule as fluorescent molecule and were able to detect the interaction between AG and SEP3 in nuclei of Arabidopsis leaf protoplasts. Techniques like this and FRET-FLIM allow the analyses of interactions between proteins in living cells, but give no information about the size of the formed complexes. To get a first indication about the stoichiometry of protein complexes, we monitored the diffusion time of in vitro synthesized AG-EYFP and SEP3-EYFP fusion proteins by means of FCS (Fluorescence Correlation Spectroscopy). From these experiments described in Chapter 6, we could speculate that SEP3 is present as a dimer and also as a higher order complex, whilst AG on its own is able to assemble into larger complexes. The diffusion time of the product formed upon co-translation of both AG and SEP3, suggests that a multimenc protein complex with a high molecular weight is formed upon interaction between AG and SEP3. Even though FCS is a powerful technique, these interpretations should be taken cautiously, mainly because these experiments were done in vitro instead of in living cells. Finally, in the last chapter we discuss the various methods that have been implemented and developed to monitor protein-protein interactions and complex formation of MADS-box transcription factors in living plant cells. Furthermore, we made a first step to monitor interactions in intact tissues under endogenous expression levels, and the preliminary results obtained from these in planta FRET-FLIM measurements are discussed.

This issue of the Journal of Fluorescence comprises a number of papers from both plenary lectures and posters presented at the Second International Fluorescence Lifetime Imaging Meeting. This series of meetings originally started as a 1-day workshop in The Netherlands in Spring 1993, with the aim of bringing together the Dutch scientists involved in fluorescence lifetime imaging and giving them a forum for exchanging ideas and presenting their latest results. The emphasis of that meeting was on new technical developments and applications in the field of fluorescence lifetime imaging. The enthusiastic reactions of the attendants of the workshop encouraged us to organize the First International Fluorescence Lifetime Imaging Meeting in Delft, which was held on November 10, 1994. The meeting was attended by over 70 scientists from different European countries and its success in terms of participants and presentations indicated the need for a periodic forum experienced by researchers in this fast developing field. Fluorescence lifetime imaging is a fast-developing interdisciplinary area of research, whereby microscopes have been adapted to use fluorescence lifetime as a contrast mechanism. This has many advantages over intensity imaging. The images obtained are independent of the variations in the fluorophore concentration in the sample. Fluorophores with overlapping emission bands can be discriminated by their different lifetimes as well as the sensitivity of the lifetime to the immediate chemical environment of the fluorophore. The technique can be implemented either in the frequency domain or using time-gating techniques, though it requires the specialization of equipment in dealing with measurements on microvolumes in the sample. A fluorescence lifetime imaging system is a synthesis of a microscope and spectroscopic measuring techniques. The main objective of the meeting was to bring together instrument developers, synthetic chemists, and fluorescence spectroscopists to interchange ideas and to stimulate the growth of this young field. The technical developments must surely be matched with the production of new fluorescent molecules which afford the selective imaging of samples of interest to physicists, chemists, and biologists. In view of the fact that all presentations contained new results, it was decided to publish them in the Journal of Fluorescence so that they will come to the attention of the wider fluorescence spectroscopy community. All papers have been peer-reviewed. We hope that in this way we will stimulate colleagues in this wide field to use the fluorescence lifetime technique for their particular applications. The editors offer their thanks to Ms. Suzy Rhinehart, Editorial Assistant to the Journal of Fluorescence, and Ms. Mary Rosenfeld, for their efforts on our behalf.

We report the simultaneous combination of three powerful techniques in uorescence microscopy: Fluorescence Lifetime Imaging (FLIM), Fluorescence Anisotropy Imaging (FAIM) and Fluorescence Recovery After Photobleaching (FRAP), also called F3 microscopy. An exhaustive calibration of the setup was carried out with several rhodamine 6G (R6G) solutions in water-glycerol and from the combination of the FAIM and FRAP data, the hydrodynamic radius of the dye was directly calculated. The F3 data was analyzed with a home-built MATLAB script, and the setup is currently explored further with Green Fluorescent Protein (GFP). Some molecular dynamic (MD) simulations are currently being run in order to help with the interpretation of the experimental anisotropy data.

Advanced non-invasive fluorescence spectroscopy and imaging for mapping photo-oxidative degradation in acrylonitrile–butadiene–styrene: A study of model samples and of an object from the 1960s

Understanding of cellular regulatory pathways that involve lipid membranes requires the detailed knowledge of their physical state and structure. However, mapping the viscosity and diffusion in the membranes of complex composition is currently a non-trivial technical challenge. We report fluorescence lifetime spectroscopy and imaging (FLIM) of a meso-substituted BODIPY molecular rotor localised in the leaflet of model membranes of various lipid compositions. We prepare large and giant unilamellar vesicles (LUVs and GUVs) containing phosphatidylcholine (PC) lipids and demonstrate that recording the fluorescence lifetime of the rotor allows us to directly detect the viscosity of the membrane leaflet and to monitor the influence of cholesterol on membrane viscosity in binary and ternary lipid mixtures. In phase-separated 1,2-dioleoyl-sn-glycero-3-phosphocholine-cholesterol-sphingomyelin GUVs we visualise individual liquid ordered (Lo) and liquid disordered (Ld) domains using FLIM and assign specific microscopic viscosities to each domain. Our study showcases the power of FLIM with molecular rotors to image microviscosity of heterogeneous microenvironments in complex biological systems, including membrane-localised lipid rafts.