Vesicle encapsulation for TIRF microscopy

The sample has to be immobilized to allow observation by single-molecule TIRF microscopy over long (seconds to minutes) timescales. To avoid artifacts from the immobilization, vesicle encapsu- lation and subsequent immobilization of the vesicles was used (Figure 4.17, Boukobzaet al., 2001; Okumus et al., 2004). Lipid films containing 300 µg 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids, Alabaster, USA) and 6 µg biotinylated lipid (1,2-Dipalmitodyl-sn- Glycero-3-Phosphoethanolamine-N-(Cap Biotinyl) (Sodium Salt)) were prepared by mixing dis- solved lipid in chloroform, evaporation of the chloroform under a stream of nitrogen and subsequent removal of residual chloroform in vacuum for at least one hour.

The lipid films were hydrated with buffer containing the sample and all other components necessary for the experiment for 30 minutes at4 ◦C. This procedure results in the formation of multilamellar vesicles (MLVs). The MLVs were then extruded 31 times through polycarbonate membranes with a pore diameter of 200 nm using an extruder (Liposofast Basic, Avestin Europe GmbH, Mannheim,

Figure 4.17:Immobilization for single-molecule TIRF experiments by vesicle encapsulation. Double-labeled Ssc1 is encapsulated together with nucleotide, substrate, or cochaperones in lipid vesicles. The vesicles are immobilized using a biotin-streptavidin-biotin linkage between biotinylated lipids in the vesicle and biotinylated PEG on the surface, and viszualized using TIRF microscopy.

Germany). This procedure breaks up the MLVs to large unilamellar vesicles (LUVs) with a homo- geneous size distribution of 200 nm diameter.

The vesicles were immobilized on a quartz prism covered with a PEG/3% Biotin-PEG layer using a Biotin-Streptavidin-Biotin linkage (Section 4.3.3). SpFRET TIRF measurements were then per- formed as described in Section 4.3.

The number of encapsulated fluorescent sample molecules per vesicle is described by a Poisson distribution. To maximize the number of vesicles encapsulating exactly one sample molecule, a sample concentration corresponding to an average of one sample molecule per vesicle (400 nM) was chosen. The number of sample molecules per vesicle was quantified in the analysis by counting the number of bleaching steps per trace. All intensity traces showing more than one distinct bleaching step were discarded from the analysis.

4.3.5 Data analysis

The raw data taken in spFRET TIRF measurements consists of movies of images taken from a single field-of-view. One half of each image consists of fluorescence in the spectral region of donor emission while the other half contains the fluorescence in the spectral region of acceptor emission.

Even if great care is taken to optically overlap the spectrally distinct images, a small shift on the order of one pixel cannot be circumvented (Figure 4.18). Complete overlap then can only be achieved by application of a coordinate transformation consisting of translation and dilation operations. The correct coordinate transformation can be derived by measurement of a sample that is clearly visible in both spectral regions at the same time, e.g. fluorescent beads with a broad emission spectrum. This allows the selection of control points in the images that can be mapped on top of each other using the MATLAB functioncp2tfrom. This transformation is then applied to extract the intensity traces from the correct transformed coordinates in the raw data movies. The coordinate transformation was applied to the image in Figure 4.18A to visualize the better overlap after the transformation (Figure 4.18B).

A peak finding algorithm now detects the positions of single molecules in the movies. Intensity time traces of donor and acceptor fluorescence are then extracted from the movie by averaging of the counts from a circular area of 3 pixel radius in each frame of the movie. The averaged counts from a ring around the molecule (3 pixel inner radius and 5 pixel outer radius) are used as background and subtracted from the intensity.

The FRET efficiencyEcan be calculated from the background-corrected fluorescence intensities of the donorIDand the acceptorIAafter donor excitation as

E = IA−CT·ID

γ·ID+IA−CT·ID

(4.45) Depending on the trace, different possibilities for determining the correction factors exist (Figure 4.19). The correction factors for crosstalk of donor fluorescence in the acceptor detection channelCT

and for different quantum yields and detection efficienciesγcan be determined directly for a specific molecule if the acceptor bleaches before the donor (Figure 4.20). The corrected total intensity has to stay constant until the donor bleaches. Thus, the average corrected total intensities before bleaching of the acceptorγhIDibef ore+hIAibef ore−CThIDibef oreand after bleachingγhIDiaf ter+hIAiaf ter− CThIDiaf ter have to be equal, hence

γ−CT = hIAibef ore− hIAiaf ter

Overlap of raw data

After image transformation

400nm

Figure 4.18:Overlay of the green and red spectral parts of one molecule. A clear shift between the detection channels exists before the application of the image transformation. After image transformation, the two peaks are perfectly overlapped.

CTcan be calculated from the intensities in the donor and acceptor detection channels after bleaching of the acceptor.

CT = hIAiaf ter

hIDiaf ter

(4.47) For molecules showing dynamic switching between states of different FRET efficiency but no ac- ceptor bleaching,γwas determined by minimizing the variance of the overall intensity. Special care has to be taken when this method is applied thatγ does not depend on the conformational state of the molecule. This can easily be the case since the quantum yields of the dyes depend on their en- vironment. For all measurements shown in this work, however, complementary MFD burst analysis measurements made it possible to rule out any dependence ofγon the conformational state.

For molecules where no direct determination of the correction factors was possible, average values forγandCTdetermined from all directly accessible values were applied.