Fluorescence microscopy

3.6. Fluorescence microscopy

In fluorescence microscopy the spontaneous light emission of a molecule is measured which is a result of a transition of an electron from a higher energy state into its initial ground state. The collapse of the molecules high energy state is responsible for the release of a photon which has a longer wavelength compared to the absorbed photon for the excitation of the molecule (Stokes-shift).¹⁰³ The absorption of a photon is very fast (10^-15 s). An average lifetime of 10^-8 s in the excited state the photon is released.

For the transition of the electron from the ground state to the electronically excited state, it is most likely that the transition occurs vertical without changes in the position of the nuclei which is called the FRANCK -CONDON principle.^{77, 103} The books “Fundamentals of Light Microscopy and Electronic Imaging” by MURPHY and “Principles of Fluorescence Spectroscopy” by LAKOWICZ give a more detailed overview to this method.^{77, 104}

Fluorescence microscopy was the main technique that was used in the experiments for this thesis, especially confocal LASER scanning microscopy (CLSM) (figure 3.20). This method enables to gather information about the geometry of adhered giant vesicles and the area change of membrane patches on a dilated PDMS surface. CLSM can be used as an imaging technique for the investigation of LUV fusion on pore spanning membranes and for the measurement of the three dimensional geometry of adhered GUVs to obtain the membrane tension of the GUVs.^{24, 49} On the one hand, in this thesis CLSM was used to measure z-stacks of GUVs to receive a three dimensional image necessary for membrane tension calculation. On the other hand, CLSM was used for the measurement of membrane areas on dilated PDMS surfaces. The used fluorescent markers were fluorescently labeled lipid dyes that are described in chapter 3.1.1. Confocal LASER scanning microscopy

Figure 3.20. Drawing of a CLSM setup.

Confocal microscopy experiments were performed with the BX61 and a FV1200 CLSM unit (Olympus, Tokio, Japan) with a 60× water objective (Olympus). In figure 3.20 the setup of a confocal microscope is drawn schematically. LASERs with a wavelength of 405 nm, 488 nm and 561 nm were used in a non-sequential mode with a scanning rate of 2 µs per pixel for each LASER. The pixel size varied but was usually kept small to reach the resolution limit of the setup.

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3.6.1. Image stacks of GUVs

Three-dimensional (3D) images were taken by changing the focal plane in z-axis (Figure 3.21) of the specimen. The collected images with a slightly moved focal plane can be stacked to generate a three-dimensional view of the adhered GUVs. Changing the focal plane was done automatically by the CLSM software on the microscope. However, the setup of the CLSM did change the focal plan in z-axis

(between 0.4-1 µm) but the received 3D image showed an elongation effect which was a general error of the CLSM setup which had to be corrected. Monodispersed glass beads and slightly adhered GUVs were used to calculate a correction factor Fcorr = Lxy / Lz for the z-stacks with the measured length Lxy in xy-plane and Lz in z-plane to receive round glass beads as can be seen from a scanning electron microscopy image in figure 3.21 A.

Figure 3.21. A) Scanning electron microscopy image of monodispersed glass beads with an average size of 6.5 µm in diameter. B) Correction factor for 3D images measured with the CLSM from image stacks of membrane coated glass beads and slightly adhered GUVs. Boxes (25%-75%), whiskers (include all points).

2 µm

A

B

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A measurement with the above mentioned CLSM of monodispersed glass spheres with a size between 3.2 µm and 7.3 µm revealed that for small image stacks an average correction factor Fcorr = 0.74 ± 0.02 (± standard deviation) for the 3D image voxel depth is needed for the correction which is shown in figure 3.21.

In figure 3.22 A the original data of a 3D image of a GUV is shown. The adhered GUV is strongly elongated and does not depict the real geometry. The average correction factor of Fcorr = 0.80 ± 0.02 for large objects like GUVs has been found during the measurements of 3D images (Figure 3.21). With this factor all image stacks of GUVs were corrected which is exemplary depicted in figure 3.22 B.

Figure 3.22. Correction of distances between focal planes for 3D images of GUVs. A) Original z-stack cross-sectional views (xz, yz). The GUV is elongated along the z-axis. B) Correction of the image stack with factor 0.8 for voxel depth led to the reduction of height and a more realistic representation of the GUV geometry.

A B

xy yz xy yz

xz yz xz

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3.6.2. Fluorescence Recovery after Photobleaching

Fluorescence Recovery after Photobleaching (FRAP) is a well-established method for the measurement of two dimensional diffusion coefficients of small molecules in thin and fluid films or lipids in membranes.¹⁰⁵ Diffusion between two membrane coated micrometer glass spheres can be measured when connected by a fusion stalk between the two membranes.²⁹ FRAP measurements therefore allowed to determine the size of the contact zone between the membrane coated spheres. The geometrical restriction of minimizing the contact zone produced a slowdown in diffusion of lipids over the hemi-fusion stalk with a recovery time between 20-30 minutes. Bleaching of a fluorescently labeled lipid dye in a SLB results in a much faster recovery of fluorescence intensity at the bleached area because of the fast and unhindered diffusion of the fluorescently labeled lipid dye from the surrounding unbleached membrane area. Typical diffusion coefficient D of fluid phase bilayers are in the range of 1 to 10 µm² s^-1.⁷⁰

FRAP provides information about the diffusion coefficient and whether the dye molecules are mobile or immobile. This information was significant because membrane fusion had to be verified by measuring the LUV dye at the SLBs. Docking of immobile LUVs on the SLBs could be partially excluded when a fast recovery of the LUV dye on the SLBs occurred after bleaching.

Experimentally, a high energy LASER pulse was used to bleach a region of interest (ROI) on the supported lipid bilayer with the actual bleaching area radius r ². After the bleaching a recovery can be detected if the fluorescently labeled lipids are mobile in the SLB. The diffusion of these lipid dye molecules from the unbleached membrane area can be measured as a fluorescent intensity curve which is shown in figure 3.19 E. Out of the recovery curve of the fluorescence intensity the half-life of recovery t_1/2 could be read. With the LASER bleaching area radius r² the diffusion coefficient D could be calculated with the following equation (14) which was found by SOUMPASIS et al.:¹⁰⁶

D=0.224∙r² t_1/2

Equation (14) was used for the analysis of FRAP measurements in this thesis to determine diffusion coefficients of the SLBs on the PDMS substrates and adhesion areas of the GUVs on functionalized glass substrates. 

In figure 3.19 an exemplary FRAP measurement is shown that depicts the bleaching and recovery of fluorescence intensity in the SLB on the PDMS surface. This FRAP-measurement was done by Jörn Dietz as a part for his master thesis. The LASER bleached a very large spot that recovers with lipid dye after a few seconds. However, the fluorescence intensity at the bleached spot did not recovered fully to its initial intensity value because of the immobile fraction of lipids in the membrane.

(14)

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Figure 3.19. Exemplary FRAP measurements on a PDMS surface fully covered with a supported lipid bilayer containing the dye TexasRed^®DHPE. A) The fluorescent images shown in a time series before and after bleaching. B) The 561 nm LASER bleached the dye lipid in the SLB in a large but defined region. C) Recovery occurs due to the diffusion of the fluorescently labeled lipids from the surrounding of the bleached area. D) After some time the recovery was finished. An immobile fraction reduces the fluorescence intensity compare to the surrounding and unbleached membrane. E) FPAP curve for the intensity of lipid dye at the bleached spot.

A B C D

E

25 µm

0 s 24 s 70 s 101 s

101

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