Skip to main content
NCBI home page
UKPMC Funders Author Manuscripts logoLink to UKPMC Funders Author Manuscripts
. Author manuscript; available in PMC: 2025 Apr 29.
Published in final edited form as: J Phys Chem Lett. 2024 Dec 5;15(50):12293–12300. doi: 10.1021/acs.jpclett.4c02731

The Molecular Mechanism of Fluorescence Lifetime of Fluorescent Probes in Cell Membranes

Yanqi Liu 1, Lydia Mathew 2, Chaofan Yu 3, Liang Fu 4, Zhengyu Shu 5,✉, Shobhna Kapoor 6,✉, Mojie Duan 7,✉
PMCID: PMC7617626  EMSID: EMS204783  PMID: 39639705

Abstract

Fluorescence probes play crucial roles in unraveling the structure and dynamics of cell membranes including membrane fluidity, polarity, and lipid molecule ordering. The fluorescence lifetime of probes describes the average duration of time that a fluorescent molecule remains in an excited state before returning to the ground state, which is sensitive to environmental changes. However, the molecular mechanism and inherent properties to determine the fluorescence lifetimes remain unexplored and inadequately studied. Furthermore, the effects of the probe on the membrane are also unclear. In this study, we investigated the interactions between probes and lipids, as well as the structural properties of probes within the outer and inner membrane of Mycobacterium smegmatis (Msm) by combining molecular dynamics (MD) simulations, enhanced sampling methods, fluorescence lifetime imaging microscopy (FLIM), and time-correlated single photon counting (TCSPC). The results show that even though the probes have very little effect on the membrane lipids, different membrane environments significantly affect the fluorescence lifetime of the probes. The analysis based on the all-atom simulations shows a strong correlation between the probe’s immersion depth within the membrane and its fluorescence lifetime. Specifically, probes buried in the membrane environment shielded from rapid water molecule collisions exhibit longer fluorescence lifetimes. The molecular basis of the fluorescence lifetime of probes in cell membranes revealed in this work would enhance the comprehension of fluorescence probes and facilitate the rational design of novel efficient probes.

graphic file with name EMS204783-f006.jpg

Open in a new tab

Cell membrane plays crucial roles in various biological processes.1 To gain deeper insights into the structure and dynamics of membrane bilayers, multifunctional and high-sensitivity detection techniques have been developed. Given that the majority of lipids inherently lack fluorescent properties, lipophilic fluorescent probes have emerged as indispensable tools, transcending the limitations posed by nonfluorescent lipids.2,3 As one of the most widely used tools, fluorescent probes provide robust support for exploring the complexities of cell membranes at the molecular level. Fluorescent probes are widely used in the studies of membrane fluidity, lipid phase state, and phase transition temperature due to their outstanding fluorescent properties and environmental sensitivity.4

Fluorescence lifetime serves as a fundamental parameter of fluorescent probes. It describes the average duration that a fluorescent molecule remains in an excited state before returning to the ground state.5,6 Minor changes in the environment, such as the polarity and viscosity, can be detected by the fluorescent probes.7,8 Even minor alterations in fluorescence lifetime can profoundly reflect the complexities of the surrounding molecular environment.9

Nevertheless, the determination factors for the fluorescence lifetimes of probes in membrane environments are obscure and are under great debate. Watanabe et al.10 used time-resolved spectroscopy (TRES) and fluorescence lifetime imaging microscopy (FLIM) to separate the long-lived and short-lived parts of fluorescent probe molecules. They suggested that water-probe collisions speed up the nonradiative relaxation process from excited states to ground states, which shortens the fluorescence lifetimes. Consequently, both the distribution and quantity of water molecules within lipid bilayers significantly affect this collisional quenching effect and the lifetime of the probes. On the other hand, the motions of the probes were believed to be the major factor deciding the lifetime of fluorescence probes. By characterizing the correlation between the probe mobility and the fluorescence lifetime with frequency-domain fluorescence measurement techniques and fluorescence anisotropy decay measurement, Parasassi et al.11 assume the rotational diffusion coefficient determines the fluorescence lifetime of probe molecules, i.e., the fluorescence anisotropy of motion-constrained probes decays slowly, resulting in longer lifetimes. Conversely, free-rotated molecules have shorter fluorescence lifetimes. Besides, the location of the probes in the membrane was proposed to be a key factor in their fluorescence lifetime. Chetan et al.12 used time-resolved fluorescence decay measurements to find that fluorescent molecules’ fluorescence lifetimes were longer in environments that were not hydrophilic. Along with the penetration into the membrane and the increasing hydrophobicity of the environment, the fluorescence lifetime of probes is increased.

In order to understand the detailed mechanism of lighting and the crucial factors to decide the lifetime of fluorescent probes, we utilized molecular dynamics (MD) simulations combined with fluorescence lifetime imaging microscopy (FLIM) and time-correlated single photon counting (TCSPC) to study the interactions between the membrane lipids and three widely used fluorescent probes, 2-dimethylamino-6-lauroylna-phthalene (Laurdan), 1,6-diphenylhexatriene (DPH), and 1-[4-(Trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH). Laurdan (Figure S1A) is a membrane-permeable fluorescent probe that exhibits spectral sensitivity to phospholipid phases in cell membranes.4,13 Laurdan prefers to immerse into its fluorophore at the glycerol backbone level of phospholipids, with its hydrocarbon tail anchored in the hydrophobic core of the bilayer, and it is sensitive to the lipid environments, such as membrane fluidity and lipid ordering.14 DPH (Figure S1B) and its trimethylammonium derivative, TMA-DPH (Figure S1C), are fluorescent probes widely used to investigate the structural and dynamic properties of cell membranes. DPH is hydrophobic and penetrates the highly disordered hydrophobic core of cell membranes.15 The fluorescence properties of DPH greatly depend on the lipid ordering; therefore, this probe is often employed for measuring membrane fluidity and lipid phase transitions.16,17 TMA-DPH anchors stably at the water/lipid interface of cell membranes,18 probing the more ordered, superficial regions of the bilayer and reflecting the fluidity state of the membrane surface.15,18

In this study, the impact of fluorescence probes on membrane properties, the insertion depth, and the molecular mechanisms of the fluorescence lifetime in cellular membranes were studied by combining computational simulations and experiments. We characterized the probes within reconstituted mycobacterial lipids extracted from the inner (IM) and outer (OM) layer of the bacterial cell envelope from Mycobacterium smegmatis (Msm), a lab model for Mycobacterium tuberculosis (Mtb).19−21 The results show that the probes have very slight effects on the lipid properties. Fluorescence polarization techniques revealed significant differences in the fluorescence lifetimes of various probes in OM and IM. The unbiased MD simulations and enhanced sampling methods give the exact locations of different probes in the membrane environments. Further, we conclude that the increase in fluorescence lifetime with deeper membrane penetration may stem from a reduction in water molecules and a decrease in collision probability.

To investigate the effect of probe binding on cellular membrane dynamics, we first analyzed the effects of probe immersion on the membrane properties, i.e., the lateral diffusion coefficients and the ordering (Scd) of lipids. We have compiled data on the lateral diffusion coefficients of various lipids and overall lipids in both the IM and OM. When there are no probes in the system (Figure 1A, Table S2), the overall lipid diffusion coefficient in IM is a little higher than that in OM. This means that lipids are more fluid in IM than in OM. In the presence of probes (Laurdan, DPH, and TMA-DPH), the overall lipid diffusion rates exhibit a slight increase for both IM and OM, demonstrating that the insertion of probes would speed up the fluidity of the membrane.

Figure 1. Effects of fluorescent probes on the membrane.

Figure 1

Open in a new tab

(A) Overall lipid lateral diffusion coefficients of different system membranes. (B) The order parameters < Scd> of lipid POPE in IM affect probes. (C) The order parameter < Scd> of lipid DPPC in OM is affected by the probe.

In addition, the effects of probe immersion on the lipid chain order parameter (Scd) were analyzed (Figure 1B, Figure 1C and Figure S4). Scd values indicate the degree of the ordered arrangement of lipid molecules. Lower Scd values mean flexible and disordered lipid tails, while higher Scd values suggest rigid and ordered lipids. Upon the introduction of probes, a slight decrease in the overall lipid Scd values for both IM and OM systems was observed, indicating a reduction in the overall lipid rigidity. Furthermore, the results show that the overall lipid order parameter in the IM system is generally higher than that in the OM system. Similar observations were reported by Tomasz et al.,22 where the fluorescent probes resulted in a slight decrease in the Scd values of phospholipids in the bilayer, indicating increased disorder, consistent with our simulation results.

By analyzing the probability of probe-lipid contact (Figure S5), we determined the binding preference of the probe for different lipids. We define the top two lipids, based on the contact probability with the probes in all lipids, as the high affinity lipids for the probes. All three probes have high affinity for CL in IM and PDIM in OM. In OM, all probes show a relatively low affinity for the lipid DPPC.

The positions of the probes in the cell membrane were calculated to define the immersion depth of probes (Figure 2 and Figure S6). The emission regions of probes such as Laurdan, DPH, and TMA-DPH mainly resides within the phenyl rings. Therefore, we used the center-of-mass (COM) of the phenyl rings to calculate the position of the probes. The results indicate that the Laurdan probe primarily localizes to the shallow regions of both the IM and OM (Figure 2A, Figure S6C, and Figure 2C), with its positioning approximately ±1.23 nm in the IM and ±2.49 nm in the OM. In contrast, although the DPH probe also demonstrates a tendency to distribute within the membrane (Figure S6A, Figure S6D, and Figure 2C), it exhibits a greater proclivity for deeply penetrating and establishing a stable presence within the central region of the lipid bilayer, occupying positions around ±0.45 nm in the IM and ±1.71 nm in the OM. Notably, the distribution pattern of the TMA-DPH probe deviates from those of Laurdan and DPH; due to the incorporation of a trimethylammonium group, it primarily congregates in the superficial layers of the membrane (Figure S6B, Figure 2B, and Figure 2C), with positioning concentrated at ±2.71 nm in the IM and ±3.14 nm in the OM, respectively. Consequently, among the three probes studied, both DPH and Laurdan predominantly localize within the membrane interior, with DPH exhibiting a preference for the central layer, whereas TMA-DPH favors the superficial regions. These findings led us to conclude that DPH and Laurdan are primarily found within the membrane interior, with DPH showing a stronger affinity for the central layer, whereas TMA-DPH tends to reside more superficially within the membrane.

Figure 2. Immersion depth of fluorescent probes in IM and OM.

Figure 2

Open in a new tab

(A) The immersion depth of Laurdan in IM as a function of simulation time. (B) The immersion depth of TMA-DPH in OM as a function of simulation time. (C) The penetration depth of three fluorescent probes in IM and OM.

The accurate position of probes in the membranes was further investigated by free energy sampling methods, which explored the free energy profiles of the probes along the reaction coordinates of the direction perpendicular to the membrane interfaces (Figures 3 and 4). The low free energy minima on the profiles according to regions where the probe molecules prefer to stay.

Figure 3. Free energy profiles of probes in IM as a function of probe position in the Z-direction (perpendicular to the membrane interface).

Figure 3

Open in a new tab

(A) Free energy profile of Laurdan in IM and the representative conformations of probes in different positions. (B) Free energy profile of DPH in IM and the representative conformations of probes in different positions. (C) Free energy profile of TMA-DPH in IM and the representative conformations of probes in different positions. In the representative structures, the lipid atoms and probe atoms are shown in balls, and the probe atoms are colored in green.

Figure 4. Free energy profiles of probes in OM as a function of probe position in the Z-direction (perpendicular to the membrane interface).

Figure 4

Open in a new tab

(A) Free energy profile of Laurdan in OM and the representative conformations of probes in different positions. (B) Free energy profile of DPH in OM and the representative conformations of probes in different positions. (C) Free energy profile of TMA-DPH in OM and the representative conformations of probes in different positions. In the representative structures, the lipid atoms and probe atoms are shown in balls, and the probe atoms are colored in green.

The results show that Laurdan exhibited three distinct states within the IM (Figure 3A). The lowest free energy minimum a located in the positions of 1.3 to 1.5 nm, indicating Laurdan prefers to immerse into the IM and align with the lipid tails. There is a high energy barrier (about 50 kJ/mol) at the center position of the membrane that obstructs the probe from crossing the membrane. The free energy profile of DPH in IM is similar to the profile of Laurdan; however, the free energy barrier is lower than that of Laurdan (Figure 3B). On the other side, TMA-DPH has a different free energy profile compared with Laurdan and DPH. The lowest free energy minimum of TMA-DPH is located at the membrane-water interface (Figure 3C), which means the probe molecule likes to stay on the membrane surface. Besides, other minima were also observed on the free energy profile of TMA-DPH, which indicates the probe has multiple binding sites in the IM.

The results indicate that Laurdan exhibits three distinct states in the OM (Figure 4A). The lowest free energy minimum, located at 1.7 to 1.9 nm, suggests that Laurdan is more likely to be embedded in the OM with its fluorescent group at the level of the phospholipid glycerol backbone, while the hydrocarbon tail is anchored in the hydrophobic core of the phospholipid bilayer. For DPH in the OM, the lowest free energy minimum is found at 0.7 to 0.9 nm, indicating that DPH is more inclined to penetrate the OM and reside in the hydrophobic core region (Figure 4B). A free energy barrier (about 13 kJ/mol) exists at the center of the membrane, hindering the probe’s passage through the membrane. The lowest free energy minimum of TMA-DPH is at the membrane-water interface (Figure 4C), suggesting that the probe molecule prefers to stay at the membrane surface. Additionally, other minima were observed in the free energy profiles of the three fluorescent probes, indicating that the probes have multiple binding sites within the OM.

To test the stability of minima observed by metadynamic simulations, we performed unbiased molecular dynamics simulations on the randomly selected conformations in the low free energy states of TMA-DPH in the IM and OM, respectively. The results show that the probe can stay in the positions of the free energy minima in the 100 ns unbiased simulations (Figure S7). Based on the unbiased MD simulations and enhanced sampling simulation results, we can conclude that both Laurdan and DPH exhibit a strong preference to immerse into the membranes (i.e., IM and OM). And the lowest free energy state of DPH is closer to the membrane center than Laurdan. Conversely, TMA-DPH shows a stronger tendency to stay at the membrane-water interface. The different positions of probes in the membrane environment might contribute to the duration of fluorescence.

In the previous sections, we studied the effects of fluorescence probes on the IM and OM of Msm membranes as well as the favorable locations of probes in the membranes by computational simulation methods. In this part, we would analyze the relationships between the fluorescence lifetime and probes/lipid properties. The simulated findings were experimentally verified by TCSPC on IM and OM liposomes generated from lipids extracted from Msm bacteria grown at 0.4 O.D. (Figure 5A and Figure S8). First, analysis of the fluorescence decay curves (Figure 5B–D) rendered lifetimes of the probes, which are the Laurdan, DPH, and TMA-DPH, by utilizing fluorescence polarization technology in the OM and IM lipid bilayers (Figure 5E). The fluorescence lifetime of Laurdan is about 5.12 ns in the IM and 2.79 ns in the OM. DPH exhibited the longest fluorescence lifetimes in both of the IM and OM; they are 9.30 ns in the IM and 6.04 ns in the OM. The fluorescence of TMA-DPH would be quenched quickly in both IM and OM; the duration time of this probe in IM is 2.30 ns and in OM is 0.62 ns.

Figure 5. Fluorescence lifetime of the probes and their correlations to the positions in the membrane.

Figure 5

Open in a new tab

(A) Visualization of phase state behavior of IM and OM using FLIM, Representative images of IM and OM (n = 16). (B)-(D) The experimental decay curves in the IM and OM layers of the probes Laurdan, DPH, and TMA-DPH, respectively. (E) Most abundant fluorescence lifetimes (<τf>) of Laurdan, DPH, and TMA-DPH in IM (white bars) and OM (gray bars) membranes. Error bars represent the standard error of the mean (SEM). (F) Correlation between the favorite positions of probes in membranes and their fluorescence lifetimes.

The experimentally measured fluorescence lifetime is highly correlated with the probe immersion depths determined by computational simulations (Figure 5F). DPH has the longest fluorescence time of the other two probes, and it is deepest buried in the membrane. On the other hand, the TMA-DPH prefers to stay in the membrane-water interfaces of the membrane, and it is the quickest quenched molecules in the three probes. Interestingly, the position of Laurdan in IM is closer to the membrane center than in OM, and its fluorescence lifetime of Laurdan in IM is also longer than in OM. The correlation coefficient of the immersed depth and fluorescence lifetime calculated based on the three probes in different membrane environments is 0.83, which demonstrated the large correlations between the probe immersed depth and fluorescence lifetime.

In order to explain the detailed mechanism of why the immersion depth decides the fluorescence lifetime of probes, we further studied the properties of probes that were affected by the location of these molecules. There are multiple factors that might be influenced by the locations; for instance, the probe rotate flexibility and the water molecule collisions were believed to have the effect of quenching the fluorescence of the probes.10,12,14 The average rotational diffusion coefficients of the probes in both the IM and the OM are given in Figure S9A. The rotational diffusion coefficient serves as a crucial parameter for characterizing molecular rotational motion and provides a clear reflection of the rotational dynamics of the probe molecules within a specific medium. The results show that the probe rotation and diffusion rates are irrelevant to the fluorescence lifetime. The correlation coefficient value of rotational rate with the probe fluorescence lifetime is 0.14, and the correlation coefficient value of lateral diffusion rate with the probe fluorescence lifetime is 0.04 (Figure S9). The probe Laurdan has slow rotation and diffusion rate in the membrane, but its fluorescence was quenched quickly. The results demonstrate that the restriction of probe motion or fluctuation is not the key factor in the long term shine of fluorescent probes.

On the other side, the locating positions of probes in the membrane will affect the water-probe interactions. If the probes are on the membrane interface or exposed in an aqueous environment, the free moving water molecules would collide with the probes and finally quench the lighting of the fluorescent probes. The deeply buried probes in the membranes would protect them from the attack of the water molecules. The in-depth analysis of the water molecule numbers in different positions of membranes shows a gradual decrease of water molecules as they close to the center of the membranes. The water molecule number at the favorable positions of the probes in different membranes is given in Figure S10. We found a strong correlation between the water molecular number and the fluorescence lifetime. The results show that the water molecules around DPH are rare (0.6 in IM and 9.5 in OM), and the average number of water molecules around Laurdan is 21.2 in IM and 89.1 in OM, respectively. In contrast, a large number of water molecules (303.3 in IM and 315.6 in OM) are present around the TMA-DPH. Based on the above results, we find that the highly mobile water molecules would collide with probe molecules, accelerating the nonradiative relaxation process from the excited state back to the ground state and thereby shortening the fluorescence lifetime. The distribution and quantity of water molecules in the lipid bilayer significantly determine the duration of the fluorescence lifetime. The deeply buried probes, which are protected by the hydrophobic environment of IM, pose longer fluorescence times.

The lipid bilayer is the fundamental structural component of the cell membrane, and observing and analyzing these lipid molecules directly through fluorescence techniques is challenging because most lipid molecules do not exhibit inherent fluorescence. To study the physical properties, dynamic behavior, and interactions of the lipid bilayer with membrane proteins, scientists use exogenous fluorescent probes. These probes can provide valuable insights into the membrane properties. It is crucial, however, to verify whether the introduction of these probes affects the membrane properties.

Plenty of previous works focused on the probe properties in different membrane environments or the membrane disturbance induced by the probe insertion, including the dynamic behavior of DPH and TMA-DPH within different membranes,23 Orlikowska-Rzeznik et al.24 explored the impact of hydration levels on Laurdan’s spectral properties, the hydration properties changes of the lipid membranes induced by the fluorescence probes,25 and the lipid properties disturbing by the probe immersions.26 Besides, many previous works studied the effects of local membrane environments to the fluorescent lifetime, for example, Ma et al. found that changes in cholesterol levels significantly affect the heterogeneity of Laurdan fluorescence lifetime in membranes.27 Bacalum et al. investigated the correlation between the generalized polarization (GP) and fluorescence lifetime in different lipid environments.28 However, the underlying factors that directly determine the lighting properties of fluorescent probes, such as the intensity and lifetime, are still unclear and poorly studied.

In our study, we selected three widely used fluorescent probes, Laurdan, DPH, and TMA-DPH, to characterize their behavior in the cell membranes of Msm. This included examining their interactions with the membranes and their effects on the membrane properties. Our results indicated that the introduction of these probes led to a slight increase in the lateral diffusion coefficients of both the IM and OM. There was also a minor decrease in lipid Scd, although the change was minimal. The three probes demonstrated a high affinity for lipid CL in the IM and lipid PDIM in the OM. The FLIM revealed significant differences in the fluorescence lifetimes of various probes in the OM and IM. Specifically, the fluorescence lifetime of the same probe was generally shorter in the OM than in the IM. Among the probes studied, DPH exhibited the longest fluorescence lifetime, followed by Laurdan, while TMA-DPH had a relatively short lifetime.

To explore these mechanisms of fluorescence lifetimes, we combined unbiased MD simulations with an enhanced sampling method to determine the positions of the probes within the membrane environment. Our atomic-level analysis of the MD simulations indicated that the increase in fluorescence lifetime could be attributed to a reduction in water molecules and a decrease in collision probability resulting from a greater membrane penetration depth. Our results resolve long-standing questions on the molecule basis of the fluorescence lifetime of probes in the cell membrane. The immersion depths of the probes are demonstrated to be highly correlated with the fluorescence lifetime in this study. Though the immersion depth of the probe in the membranes is a crucial property, it is hard to directly observe by “wet” experiments. Computational simulations provide a potential way to determine that. However, the conventional all-atom simulations are not able to obtain accurate probe positions due to the unaffordable computational costs. In this study, by employing advanced enhanced sampling methods, we characterized the accurate locations and immersion depths of the probes in membranes for the first time. In addition, we investigated the fluorescent probes in the highly complex natural membranes from Mycobacterium smegmatis, which is a causative agent of Tuberculosis (TB). Our study shows significant potential to study the complex mycobacterial membrane biophysics by using well-characterized aspects of lipid probes. By elucidating how the fluorescence lifetime varies with membrane penetration depth, our study uncovers the intrinsic mechanisms behind these variations and provides the foundation for optimizing fluorescent probes.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpclett.4c02731.

Methodology, simulation time table, lipid properties table, molecular structure figure, simulation relevant figures (PDF)

Transparent Peer Review report available (PDF)

Supporting Information

Acknowledgments

This work is supported by the National Natural Science Foundation of China (21773298) to M.D., the DBT/Welcome Trust India Alliance Fellowship (IA/I/21/1/505624) to S.K., and the National Natural Science Foundation of China (31370802) to Z.S.

Footnotes

Notes

The authors declare no competing financial interest.

Contributor Information

Yanqi Liu, College of Life Science, Fujian Normal University, Fuzhou 350117, China; NMR and Molecular Sciences, School of Chemistry and Chemical Engineering, The State Key Laboratory of Refractories and Metallurgy Wuhan University of Science and Technology, Wuhan 430081, China.

Lydia Mathew, Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India.

Chaofan Yu, College of Life Science, Fujian Normal University, Fuzhou 350117, China.

Liang Fu, NMR and Molecular Sciences, School of Chemistry and Chemical Engineering, The State Key Laboratory of Refractories and Metallurgy Wuhan University of Science and Technology, Wuhan 430081, China.

Zhengyu Shu, College of Life Science, Fujian Normal University, Fuzhou 350117, China.

Shobhna Kapoor, Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India.

Mojie Duan, NMR and Molecular Sciences, School of Chemistry and Chemical Engineering, The State Key Laboratory of Refractories and Metallurgy Wuhan University of Science and Technology, Wuhan 430081, China.

References

  • (1).Gennis RB. Biomembranes: Molecular Structure and Function. Springer; New York: 2013. [Google Scholar]
  • (2).Demchenko AP, Mély Y, Duportail G, Klymchenko AS. Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes. Biophys J. 2009;96:3461–3470. doi: 10.1016/j.bpj.2009.02.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (3).Klymchenko AS, Kreder R. Fluorescent probes for lipid rafts: from model membranes to living cells. Chemistry & Biology. 2014;21:97–113. doi: 10.1016/j.chembiol.2013.11.009. [DOI] [PubMed] [Google Scholar]
  • (4).Sánchez SA, Tricerri M, Gunther G, Gratton E. Laurdan generalized polarization: from cuvette to microscope. Modern Research and Educational Topics in Microscopy. 2007;2:1007–1014. [Google Scholar]
  • (5).Shinitzky M, Barenholz Y. Fluidity parameters of lipid regions determined by fluorescence polarization. Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes. 1978;515:367–394. doi: 10.1016/0304-4157(78)90010-2. [DOI] [PubMed] [Google Scholar]
  • (6).van der Meer BW, van Hoeven RP, van Blitterswijk WJ. Steady-state fluorescence polarization data in membranes. Resolution into physical parameters by an extended Perrin equation for restricted rotation of fluorophores. 1986;854:38–44. doi: 10.1016/0005-2736(86)90062-3. [DOI] [PubMed] [Google Scholar]
  • (7).Peng X. Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor for dual mode imaging of cellular viscosity. Journal of the American Chemical Society. 2011;133:6626–6635. doi: 10.1021/ja1104014. [DOI] [PubMed] [Google Scholar]
  • (8).Ragaller F, et al. Quantifying Fluorescence Lifetime Responsiveness of Environment-Sensitive Probes for Membrane Fluidity Measurements. J Phys Chem B. 2024;128:2154–2167. doi: 10.1021/acs.jpcb.3c07006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (9).Li L-L. BODIPY-based two-photon fluorescent probe for real-time monitoring of lysosomal viscosity with fluorescence lifetime imaging microscopy. Anal Chem. 2018;90:5873–5878. doi: 10.1021/acs.analchem.8b00590. [DOI] [PubMed] [Google Scholar]
  • (10).Watanabe N, Suga K, Slotte JP, Nyholm TK, Umakoshi HJL. Lipid-surrounding water molecules probed by time-resolved emission spectra of laurdan. Langmuir. 2019;35:6762–6770. doi: 10.1021/acs.langmuir.9b00303. [DOI] [PubMed] [Google Scholar]
  • (11).Parasassi T, De Stasio G, Rusch RM, Gratton EJBj. A photophysical model for diphenylhexatriene fluorescence decay in solvents and in phospholipid vesicles. Biophys J. 1991;59:466–475. doi: 10.1016/S0006-3495(91)82240-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (12).Poojari C, et al. Behavior of the DPH fluorescence probe in membranes perturbed by drugs. Chemistry and Physics of Lipids. 2019;223:104784. doi: 10.1016/j.chemphyslip.2019.104784. [DOI] [PubMed] [Google Scholar]
  • (13).Weber G, Farris FJ. Synthesis and spectral properties of a hydrophobic fluorescent probe: 6-propionyl-2-(dimethylamino) naphthalene. Biochemistry. 1979;18:3075–3078. doi: 10.1021/bi00581a025. [DOI] [PubMed] [Google Scholar]
  • (14).Parasassi T, Krasnowska EK, Bagatolli L, et al. Laurdan and Prodan as polarity-sensitive fluorescent membrane probes. Journal of Fluorescence. 1998;8:365–373. doi: 10.1023/A:1020528716621. [DOI] [Google Scholar]
  • (15).Cundall R, Johnson I, Jones M, Thomas E, Munro I. Photophysical properties of DPH derivatives. Chem Phys Lett. 1979;64:39–42. doi: 10.1016/0009-2614(79)87270-X. [DOI] [Google Scholar]
  • (16).Lentz BR. Use of fluorescent probes to monitor molecular order and motions within liposome bilayers. Chem Phys Lipids. 1993;64:99–116. doi: 10.1016/0009-3084(93)90060-g. [DOI] [PubMed] [Google Scholar]
  • (17).Lentz BR. Membrane “fluidity” as detected by diphenylhexatriene probes. Chemistry and Physics of Lipids. 1989;50:171–190. doi: 10.1016/0009-3084(89)90049-2. [DOI] [Google Scholar]
  • (18).Prendergast F, Haugland R, Callahan PJB. 1-[4-(Trimethylamino) phenyl]-6-phenylhexa-1, 3, 5-triene. synthesis, fluorescence properties and use as a fluorescence probe of lipid bilayers. 1981;20:7333–7338. doi: 10.1021/bi00529a002. [DOI] [PubMed] [Google Scholar]
  • (19).Adhyapak P, et al. Dynamical organization of compositionally distinct inner and outer membrane lipids of mycobacteria. Biophys J. 2020;118:1279–1291. doi: 10.1016/j.bpj.2020.01.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (20).Bansal-Mutalik R, Nikaido H. Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides. Proc Natl Acad Sci USA. 2014;111:4958–4963. doi: 10.1073/pnas.1403078111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (21).Adhyapak P, et al. Lipid clustering in mycobacterial cell envelope layers governs spatially resolved solvation dynamics. Chem Asian J. 2022;17:e202200146. doi: 10.1002/asia.202200146. [DOI] [PubMed] [Google Scholar]
  • (22).Róg T, Girych M, Bunker AJP. Mechanistic understanding from molecular dynamics in pharmaceutical research 2: lipid membrane in drug design. Pharmaceuticals. 2021;14:1062. doi: 10.3390/ph14101062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (23).do Canto AMTM, et al. Diphenylhexatriene membrane probes DPH and TMA-DPH: A comparative molecular dynamics simulation study. 1858. 2016;11:2647–2661. doi: 10.1016/j.bbamem.2016.07.013. [DOI] [PubMed] [Google Scholar]
  • (24).Orlikowska-Rzeznik H, Krok E, Chattopadhyay M, Lester A, Piatkowski L. Laurdan Discerns Lipid Membrane Hydration and Cholesterol Content. J Phys Chem B. 2023;127:3382–3391. doi: 10.1021/acs.jpcb.3c00654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (25).Watanabe N, et al. Solvatochromic Modeling of Laurdan for multiple polarity analysis of Dihydrosphingomyelin bilayer. Bio-physical Journal. 2019;116:874–883. doi: 10.1016/j.bpj.2019.01.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (26).Suhaj A, Gowland D, Bonini N, Owen DM, Lorenz CD. Laurdan and Di-4-ANEPPDHQ Influence the Properties of Lipid Membranes: A Classical Molecular Dynamics and Fluorescence Study. J Phys Chem B. 2020;124:11419. doi: 10.1021/acs.jpcb.0c09496. [DOI] [PubMed] [Google Scholar]
  • (27).Ma Y, Benda A, Kwiatek J, Owen DM, Gaus K. Time-resolved laurdan fluorescence reveals insights into membrane viscosity and hydration levels. Biophysical Journal. 2018;115:1498–1508. doi: 10.1016/j.bpj.2018.08.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (28).Bacalum M. Generalized polarization and time-resolved fluorescence provide evidence for different populations of Laurdan in lipid vesicles. Journal of Photochemistry and Photobiology B: Biology. 2023;250:112833. doi: 10.1016/j.jphotobiol.2023.112833. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
EMS204783-supplement-Supporting_Information.pdf (732.6KB, pdf)

RESOURCES