Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored Using Electrophoresis and Fluorescence Lifetime Imaging Microscopy

Abstract

Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can undergo "self-quenching" of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artifacts in fluorescence images and relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores associated with supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of lipid-linked Texas Red (TR) fluorophores were generated within 100 × 100 μm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively charged TR-lipid molecules toward the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mol/mol), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mol/mol), leading to the reduction of fluorescence lifetime down to 30% and quenching of the fluorescence intensity down to 10% of their original levels. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The calculated concentration profiles have a good fit to an exponential growth function, suggesting that TR-lipids can diffuse freely even at high concentrations. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.

Figure 1

Concept for “in-membrane electrophoresis” experiments. (A) Example fluorescence microscopy image of the template pattern of photopolymerized DiynePC lipids (1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine), with excitation at 485 nm and collection of emission between 505 and 535 nm. These templates were generated in a microarray pattern by UV exposure through a photomask (see the Experimental Methods section for details). (B) Chemical structure of the fluorescent lipid TR-DHPE (Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine). Red circles and blue circles mark positive and negative charges, respectively. (C) Chemical structure of the lipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). (D) Schematic of a lipid bilayer confined by the barriers of the DiynePC template (black). In the absence of any electric field, TR fluorophores (red) will be uniformly distributed in this membrane corral, with a screen of ions (purple) close to the membrane surface. (E) As in (D) but with an applied E-field. (F) Schematic of the electrophoresis flow cell (not to scale), key parts labeled.

Figure 2

Kinetic analysis of the electrophoretic migration and fluorescence quenching of TR. (A) Time-lapse series of fluorescence intensity images of a lipid bilayer corral containing 0.28% (mol/mol) TR-DHPE after commencing the application of an electric field (45 V/cm). Each image represents a 5-frame acquisition (16 s exposure) at the standard excitation laser power (see Methods). (B) Average intensity profiles measured in the red, dashed box region in panel (A). Black-to-red lines represent increasing time points in a range from 0 to 192 s separated by 16-s intervals. Inset: The midpoint (half-maximum intensity) of the “moving edge” of fluorescence at the right side of the corral is measured for each time point (blue circle). (C) Graph showing the displacement of the moving edge measured in (B) with increasing time points. The drift velocity, V_drift, is obtained from a linear fit (blue line). (D) Time-lapse series of FLIM images, as in (A) except including a color-scale for fluorescence lifetime. (E) Fluorescence decay curves obtained by accumulating photons collected at the edge of the bilayer (white dashed regions in (D)) after the application of the E-field, at the time points noted. (F) The fitted lifetime, ⟨τ⟩, at the edge of the corral as analyzed in (E), plotted against time after the application of the E-field.

Figure 3

Demonstration of the method for generating concentration profiles from a FLIM image. (A) Example FLIM images of a sample series of SLBs containing TR at defined concentrations, as labeled (each image: 25 frames at standard laser power). (B) Graph showing the raw fluorescence intensity (open red squares) and the fluorescence lifetime (blue squares), as calculated from the average of all pixels in an image. The corrected fluorescence intensity, F₀ (filled red squares), was calculated using eq 1 as described in the text, and the tabulated results are shown in Table S2 in the Supporting Information. The solid red line is a linear fit, F₀ = mC + Y₀, with the solution m = 168.3 and Y₀ = 10.7. The fact that Y₀ is not equal to zero is likely to be due to a low amount of fluorescence background. (C) Example FLIM image of a 0.28% (mol/mol) TR corral at equilibrium in an E-field. The white dashed box denotes the ROI from which horizontal profiles of lifetime and intensity were obtained by averaging the pixels accumulated vertically (typically 150 pix/vertical, improving the signal-to-noise). (D) Profile of fluorescence lifetime against the x-position obtained from the white dashed region in (C). (E) Profile of the raw fluorescence intensity profile (light red), F, obtained from the white dashed region in (C). The nonquenched intensity, F₀ (dark red), was calculated from the profile for F(x) and the profile for τ(x) using eq 1 in the following form: F₀(x) = F(x)·e^{2 ln[τ₀/τ(x)]}. (F) The concentration profile (blue) calculated from the data for F₀ from (E) using the direct proportionality relationship between the molar concentration and nonquenched intensity of a fluorophore, C = (F₀ – 10.7)/168.3. The solid black line is a fit to the monoexponential function: C(x) = a·e^–bx + y₀ (a, b, and y₀ are fitting constants).

Figure 4

Comparison of the consistency of fluorescence lifetime profiles and calculated concentration profiles for multiple corrals within one sample. (A) FLIM images of six different corrals at equilibrium during electrophoresis (45 V/cm) (each image: 25 frames at standard laser power). This sample had a starting concentration of 0.28% mol/mol TR-DHPE to DOPC. The colored dashed boxes show the regions-of-interest from which lifetime and concentration profiles were obtained (as described in Figure 3). Different colored dashed boxes correspond to the different colored scatter plots in subsequent panels. (B) Multiple profiles of fluorescence lifetime vs x-position across the membrane corral, corresponding to the ROIs indicated in (A). (C) Multiple profiles of raw fluorescence intensity vs x-position. (D) Multiple profiles of the calculated concentration profiles, generated as described in Figure 3. The black line shows a fit of the combined dataset to a monoexponential function: C(x) = a·e^–bx (a and b are fitting constants).

Figure 5

Comparison of the fluorescence properties of membrane corrals before and after electrophoresis for SLBs containing a range of starting concentrations of TR. (A) FLIM images of corrals containing either 0.28, 0.57, or 0.85 mol/mol % TR (relative to DOPC) before application of an E-field. (B) FLIM images of the corrals from (A) at equilibrium during electrophoresis (45 V/cm). The white arrow indicates a defect in the SLB that appeared during electrophoresis, and the related area (circled in white) was excluded from later analyses. (C) Multiple profiles of fluorescence lifetime vs x-position across the membrane corral, corresponding to the ROIs indicated in (B). The mean lifetime was used for this analysis, and for completeness, the multiexponential character of the fluorescence decay curves was analyzed elsewhere (see Figure S5). (D) Multiple profiles of raw fluorescence intensity vs x-position. (E) Multiple profiles of the corrected fluorescence intensity (right axis) and the equivalent calculated concentration (left axis), generated as described in Figure 3.

Figure 6

Assessing whether concentration profiles follow a monoexponential function. (A) Selected concentration profiles as a scatter plot, colored as in Figure 5. The black lines are a fit to a monoexponential function: C(x) = a·e^–bx (a and b are fitting constants), performed for each profile. (B) The same data and fitting as in (A), except displayed as a semilogarithmic plot.