Real-Time Monitoring of Protein Binding and Imaging Lipid Domains with Surface Enhanced Ellipsometry Contrast (SEEC) Microscopy

Topics Covered

Experimental Setup
Real-time Recording of Label-free SLB Formation
Label-free Imaging of Spatially Heterogeneous SLBs
Label-free Imaging of Spatially Heterogeneous SLBs
Advantages of SEEC Microscopy


Surface enhanced ellipsometry contrast (SEEC) microscopy can be used for label-free imaging of spatially heterogeneous supported lipid bilayers (SLB) or lipid domains. Furthermore, time-lapse imaging enables time-resolved, label-free visualization of biomolecular recognition events on these heterogeneous SLBs.

Protein binding events can be monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ∼1 Hz with a sensitivity in terms of surface coverage of ∼1 ng/cm2.

Hence, despite the significant improvement in spatial resolution compared with alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance imaging (SPRi) and imaging ellipsometry.

In this study by Dr A. Gunnarsson and Prof. F. Höök of the Chalmers University of Technology, Sweden, in collaboration with Nanolane, SEEC microscopy was used to discriminate local (sub-μm scale) differences in protein binding by time-resolved imaging of anti-GalCer antibodies binding to phase separated lipid bilayers consisting of phosphatidylcholine (POPC) in the fluid phase and galactosylceramide (GalCer) in the gel phase.

To support the data interpretation, the advantage of SEEC imaging to allow direct complementary information by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes is also demonstrated.

By operating SEEC microscopy on an inverted optical microscope equipped with a TIRF illuminator, the illumination conditions can be modified closer to the optimal lighting conditions (annular aperture instead of a conic one).

This leads to highly contrasted images (even with high magnification objectives) and thus, a clear distinction between the two lipid phases despite a thickness difference of only ∼1 nm.

Experimental Setup

All micrographs were acquired with an inverted microscope (Nikon Ti Eclipse, Japan) equipped with a 60× oil immersion objective (NA = 1.49) and a cooled EMCCD camera (iXon, Andor Technology, N. Ireland) at an image frame rate of 12 or 20 images/min.

The microscope was equipped with a perfect focus system (PFS) to compensate for focus drift over time with an accuracy of ±25 nm. The samples are illuminated with light at 525 nm, close to the optimal settings (550 nm) for the SEEC substrates.

The first of the two required polarizers was placed at the entrance of the TIRF illuminator (TI-SFL Super Epi-Fluorescence illuminator, Nikon, Japan) and the second polarizer was placed below the filter turret and adjusted to be orthogonal to the first polarizer by minimizing the reflected intensity from the surface.

The TIRF illuminator was used to adjust the angle of the incident light to yield a good contrast between the surface and the deposited film (Fig. 1). The range of angles which fulfilled this condition was estimated to be somewhere in the interval of (25°-50°) for the SEEC substrates.

Figure 1. Schematic illustration of the aperture in the TIRF illuminator used to select a small range of angles of incidence for TIRF (typically ~65°) and for SEEC (25°-50°).

Real-time Recording of Label-free SLB Formation

First, it has been demonstrated that SEEC has the capability to image, in real-time, the deposition of lipid vesicles which leads to the spontaneous rupture and subsequent fusion of bilayer patches into a continuous SLB on SiO2.

Using a microfluidic channel with four channel arms in a cross channel geometry, POPC vesicles were injected against a counter flow of buffer such that the lipid vesicles were only allowed to adhere to and subsequently rupture on a predefined region of the SiO2-coated SEEC substrate.

In this way, an unaffected area of the substrate could be imaged simultaneously with the SLB formation process (Fig. 2), thus acting as a reference area. The reference area provides a means to compensate for focus drift and intensity fluctuations in the illumination that may otherwise influence the recorded change in intensity during time-resolved measurements.

Note that besides a gradual increase in the optical contrast as more lipid material is deposited onto the surface, the contrast exhibits a smoothening upon SLB formation. This observation is attributed to the conversion of discrete lipid vesicles into a continuous lipid bilayer film.

Figure 2. Top: Inverted SEEC micrograph snapshots of the SLB formation process upon injection of unlabeled POPC vesicles in a microfluidic cross channel with an opposite flow of buffer. Scalebar is 50μm. Red curve show the intensity profile across the SLB edge averaged over 100μm. Bottom: 3D illustration of the same micrographs.

Label-free Imaging of Spatially Heterogeneous SLBs

Second, SEEC microscopy can image, without the use of labels, a spatially heterogeneous SLBs consisting of a phase separated POPC (in the fluid phase) and GalCer (in the gel phase).

While the phase-separated SLB cannot be visualized using ordinary bright-field illumination (inset, Fig 3), the enhanced contrast of SEEC enables a clear visualization of the domains despite a thickness difference of only ∼1 nm.

Figure 3. SEEC micrograph of phase separated GalCer-rich gel phase domains (dark), surrounded by POPC-rich fluid phase (bright area). Red curve show intensity profile across the domain edge. Scale bar is 20μm. Inset: Bright-field micrograph of the same surface.

Label-free Imaging of Spatially Heterogeneous SLBs

Finally, it has ben shown that SEEC has the capability to monitor time-resolved biorecognition events on these heterogeneous SLB in a label-free manner.

Protein binding can be monitored with a lateral resolution near the optical diffraction limit and hence one can easily distinguish the binding pattern on the lipid gel and fluid phase separately (red and blue binding curve).

All micrographs are divided with the first frame of the measurement (prior to protein injection) to isolate contrast changes due to protein binding, as seen in Fig. 4.

Figure 4. Right: Binding curves, represented by a decrease in intensity upon binding of anti-GalCer antibodies to the gel phase domains (red) and surrounding fluid phase (blue) including negative controls using anti-IgG antibodies on identical phase separated SLBs (magenta) and anti-GalCer antibodies on pure POPC SLB (cyan). Left: SEEC micrograph snapshots during protein binding. Micrograph are divided by the first frame (Fig. 3) to isolate contrast changes due to protein binding.

Advantages of SEEC Microscopy

  • Label-free imaging of protein binding in real-time with diffraction-limited resolution
  • Direct (in situ) compatibility with fluorescence microscopy (incl. TIR, FRAP, FRET...)
  • Compatibility with microfluidic applications

Source: Nanolane

For more information on this source please visit Nanolane

Date Added: Mar 5, 2013 | Updated: Mar 5, 2013
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