Lipid bilayer conjugated with a fluorophore on a plasmonic surface has been investigated to understand the photostability and localization of a single molecule on a 2D surface. This paper focuses on the longer photostability of a fluorophore, which has been used to develop better in-plane resolution platform to study the localization of a fluorophore on a 2D bilayer system using a hand-made total internal reflection fluorescence microscopy i.e. TIRF (see Fig. 1a). The blinking information from each single molecule (as shown in Fig. 1b) has further been analyzed. An optimum thickness of 20 nm that includes a lipid bilayer film and a plasmonic surface has been standardized to achieve better photostability (data not shown). The lipid bilayer serves as a spacer layer in between plasmonic surface and the fluorophore.
Localized surface plasmon resonance enhancement of the electromagnetic fields of a few nanometer thick fluorophore conjugated lipid bilayer film deposited on a 1 nm Au thin film was observed. The thickness of the gold film (see Fig. S1 and Fig. S2) was optimized because the more the overlap in between the surface plasmon resonances of the plasmonic film with the emission of Alexa 488, the more the photostability can be achieved. A series of thickness of Au film was investigated (Fig. 1c) and found that 1 nm Au film results better photostability and that is because of maximum overlap in between the surface plasmon resonances of the plasmonic film with the emission of Alexa 488. Experiment was further carried out for longer time to understand the plasmonic effect on photostability in a longer time period (Fig. 1c). Even after 45 minutes the intensity of Alexa 488 covalently attached with lipid bilayer surface deposited on top of a 1 nm gold surface didn’t decrease (Fig. 1c, red color) much whereas it was hardly be seen on just top of a glass surface (Fig. 1c, black color). The dependence of thickness of the plasmonic surface was investigated, and it was observed that the photostability decreased with increase in thickness of the plasmonic film (results are shown in Fig. 1c). The thicker plasmonic film (2 nm Au film and 4 nm Au film) quenches the fluorescence intensity of the fluorophore. It was also observed that the photostability decreased asymptotically with increase of the thickness of Au film (Fig. 1c, green color and Fig. 1c, purple color). Almost a factor of fourty in photostability was achieved using a 1nm gold film. To further confirm the importance of the plasmon peak position in achieving photo stability enhancement, alexa 488 embedded bilayer was deposited onto a glass substrate on top of a 1 nm thick film of copper (Cu). The plasmon of Cu absorbs at lower energy and does not spectrally overlap well with the emission of alexa 488 and resulted less photostability (Fig. 1c, blue color). These results confirm that good spectral overlap of the molecular emission with the plasmon absorption is required to achieve enhanced photo-stability. In addition to the photo-stability enhancement brought about through plasmonic interactions (Fig. 2a), single molecules of alexa 488 show significantly reduced fluorescence intermittency on the Au substrate (Fig. 2b). The ‘on-off’ emission switching that occurs on the timescale of seconds is because of the reversible formation of a polaron due to energy release of an electron to aerial oxygen. The fluorescence lifetimes were measured (Fig. 2c). Alexa 488 single molecules exhibit single exponential decay kinetics on glass (lifetime 3.8 ns). On 2nm Au, the decay rate is similar to the glass but on 1nm Au the decay rate is much faster (lifetime 1.2 ns). The observed decrease in polaron formation and less photo-degradation on 1 nm Au film are both consistent with a reduction in the excited state lifetimes due to plasmonic interactions.
Further, second order co-relation measurements were carried out to confirm the single molecule study. Organic molecules pumped at low excitation rates, means that there is one excitation and emission event within a single laser pulse. This relaxation time is governed by the triplet lifetime. It is also possible to observe two emission events within a single laser pulse if; the ground state is re-populated due to rapid radiative decay. g2 (τ) showed the anti-bunching behavior which demonstrates that the predominant emitters are single molecules rather than aggregates (see Fig. 2d). We performed the experiment from bulk and gradually decreasing the concentration of the fluorophore to achieve single molecule. We observed bunching at bulk and also for the aggregates (the black and purple color in Fig. 2d confirmed it) and finally we got anti-bunching (as shown in blue color in Fig. 2d). The value was found to be 0.3.
The rigidity of the bilayer (see Fig. S3-Fig. S5) was further changed so that we can track the lateral translocation of a single fluorescent molecule. The molecular positions of the molecule were extracted from the videos captured at single molecule level. Under ideal conditions, the average intensity produced by a single emitter is proportional to the cross-section of the microscope’s point spread function (PSF).
I(x^',y^' ) = H(x,y,z,Ns,Nb) = H(θ); Where x’, y’ the positions in the image plane when the emitter is at position x, y, z in the sample space, where θ represents these position and photon parameters; Ns is the total signal photons and Nb is the constant background per unit area. The actual image from the detector is subject to noise and binned into pixels, producing a signal nk at each of the pixels wk within the region of interest (ROI).
Therefore, the position estimator can be defined by
x^''=(∑nk.wk )⁄(∑nk)
The imaging model H (θ) produces an expected value of the image, I (x’, y’; θ), and the best guess of θ is found by optimizing I to match the observed PSF. LS was one of the first relatively unbiased localization algorithms used (26, 27) and is familiar from its ubiquitous application to scientific regression problems and curve fitting. The scoring method of LS, (27, 28) is to minimize the square error between the PSF model u (θ) and the observed data can best be described by
S=∑[ nk-uk(θ)]2
When molecules emit light isotopically, without net polarization, the scalar approximation allows for a comprehensive treatment of the imaging system The image of an ideal, in-focus emitter at the coverslip can be best described by
I(x^',y^' ) = C[(J(kNAρ))⁄(kNAρ]2)
where J being the first-order Bessel function of the first kind, k the wavenumber 2π/λ, ρ the distance from the point source in the image plane and C a constant for a given number of total detected photons. As the PSF’s intensity is concentrated in the center, therefore, a Gaussian function is a tractable and reasonably accurate approximation, and it is extremely common to fit single-molecule data with a symmetric Gaussian plus a constant background with the width σ the size of the spot arising from the diffraction limit (27, 28)
I(x^',y^' ) = Ns/2πσ2 exp (-ρ2/2σ2) + Nb
Based on this model a position of the conjugated fluorophore was estimated close to that of the true position that avoids the bias of the simpler estimators. But the essential question is how precise an individual localization is. To address this, we can either simulate or measure the spread of position estimates obtained from the same molecule emitting over many frames. Repeated least-squares localizations on simulated data give a range of estimates with standard deviation, or in other words, the localization precision can be achieved. But again, the localization precision is dependent on the estimator also. Therefore, the correction for localization precision (27, 28) which is best described by the proposed equation as described below.
σ2=(σi2 + a2/12)/Ns(16/9 + 8πNb(σi2 + a2/12)/(Ns.a2); Where standard deviation σi detected with pixels of area a2 and gives the correct result with a low background and high intensity i.e., due to presence of surface plasmon, and concisely predicts the precise localization. Based on this model, a strategy has been proposed to understand the lateral localization of single molecule on a 2D surface (scheme has been provided in Fig. 3a). Histograms of standard deviations of localizations from single molecules in x and y has been shown in Fig. 3b.
It's clear seen that when the labeled lipid concentration is lower the average mean free path between labeled lipids increases (Fig. 4a), and that's why the distribution of pixel intensity is getting decreased (Fig. 4b). In other words, bilayers with higher lipid concentration are more constrained, and therefore, the diffusion is rather more restricted (as shown in Distance vs. Time graph in Fig. 4c and Fig. 4d) and that's why the distribution of average intensity doesn't change significantly within the ROI. Based on this information further simulation has been carried out.
A single Alexa 488 molecule embedded on a lipid bilayer deposited on a 1 nm gold surface was horizontally moved to test the ability to measure the thickness of the plasmonic surface precisely with the step sizes of the heterostructure. Fluorescent images were collected with 0.5-s integration time. Several individual spots (optimized with the maximum pixel intensity as shown in Fig. 5a), corresponding to different concentration of Alexa 488 conjugated bilayer and approximately 5,000 to 10,000 photons per spot per image were collected, enabling us to locate the center to within the range of 20 nm to 80 nm typically, and, for brighter spots, 40 nm (see Fig. 5b).
