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All images were 8-bit-encoded digital images acquired on a Leica TCS SP2 AOBS confocal microscope (Leica). Live cell imaging was carried out with a micro Live Cell Incubation System (H.Saur, Reutlingen, Germany). To avoid cross talk or bleed through in dual and multiple color imaging, a sequential scanning method was employed so that all the emission spectra are sufficiently separated from each other at the initial step of image acquisition. The sequential

Materials and methods

acquisition method was performed by exciting only one fluorophore at a time, and detecting within the range of the emission spectra of the fluorophore concomitantly.

3.8.2 Colocalization analysis by confocal microscopy

For the purpose of colocalization analysis, confocal images were acquired with an aqueous immersion objective (63×, NA 1.2) in live cell studies. A sequential acquisition method was usually applied in order to unambiguously distinguish the different emission spectra of different fluorophores in the sample. Selected dual or triple color composite images were analyzed with the Leica Confocal Software (LCS, Leica) for colocalization of different fluorophores. This digital analysis of colocalization of two or more fluorescent molecules provides information as to whether the fluorescence signals occupy the same pixel in the analyzed image. Compared to commonly used ‘visualized colocalization’ by simple overlapping, the digital colocalization analysis presents precise spatial localization and accurate fluorescence intensities of individual fluorescent molecules at each pixel in the entire field of recording. The colocalization measurement performed in this work is based on a statistical approach that performs intensity correlation coefficient-based (ICCB) analysis. In brief, the pixel grey values of each fluorophore in the image are plotted against each other and displayed in a pixel distribution diagram, called the fluorogram. For each given pixel in the multicolor image, the intensity value of fluorophore 1 is used as the x-coordinate and the intensity value of fluorophore 2 (and fluorophore 3, if required) as the y-coordinate (and the z- coordinate) of a two-dimensional (or three-dimensional) fluorogram. Thus the dimmer pixels in the image are close to the origin of the fluorogram, while the brighter pixels are farther out. The pixels with only one fluorescence signal are clustering toward the axes of the fluorogram, and the pixels with both (or all the three) fluorescence signals are clustering toward the centre of the fluorogram. Those clusters of pixels with fluorescence signals are called ‘clouds of signals’ below. Taking advantage of digital analysis, the colocalizing or non-colocalizing pixels were replotted for visualization of the original spatial information, as only those selected pixels are presented in the

In addition to the commercial software (LCS, Leica), a set of MatLab programs were written for special colocalization analysis in this work. In brief, these programs offer the possibility to analyze multicolor xyzt four-dimensional image series, as well as determine the threshold values for each channel in each image according to the factor of photobleaching caused by long-term image acquisition. More details, including the theoretical background and formulas, can be found in Chapter 4.2.4.4.

3.8.3 Fluorescence Resonance Energy Transfer (FRET) by confocal microscopy

FRET, also known as Förster Resonance Energy Transfer, is a photophysical phenomenon involving the radiationless energy transfer from a donor fluorophore to an appropriately positioned acceptor fluorophore (Förster, 1948; Förster, 1965; Herman, 1989; Stryer, 1978; Van Der Meer et al., 1994; Wu and Brand, 1994). FRET occurs only if the spectral, dipole orientation, and distance criteria of both donor and acceptor fluorophores are satisfied. The efficiency of resonance energy transfer is defined by the formula

E = R06 / (R06 + r6)

where r is the distance between the two fluorophores, and R0 is the distance at which 50% energy

transfer takes place.

Excitation of the donor fluorophore results in quenching of donor emission and increased, sensitized acceptor emission. Intensity-based FRET measurement techniques are based on these effects, which have been applied in this work for detection of protein-protein interaction in living cells by confocal microscopy. In brief, nine 12-bit-encoded digital images are required for an analysis by the sensitized acceptor emission method, including donor only, acceptor only, and double-labeled samples for three channel-imaging: donor emission upon donor excitation (donor channel), acceptor emission upon acceptor excitation (acceptor channel), and acceptor emission upon donor excitation (FRET channel). The approach is based on the assumption of same spectral

Materials and methods

conditions. The formula used to calculate true FRET values and FRET efficiencies (FRETeff) are shown below:

FRET = B – b * A – (c – a * b) * C

FRETeff = FRET / C

where

A = channel 1 = Donor emission (by excitation of the donor) B = channel 2 = FRET emission (by excitation of the donor) C = channel 3 = Acceptor emission (by excitation of the acceptor) A, B, and C are background corrected 12 bit values.

a = Correction factor of acceptor only measurement

= Donor emission (by excitation of the donor) / acceptor emission (by excitation of the acceptor) b = Correction factor of donor only measurement

= Acceptor emission (by excitation of the donor) / donor emission (by excitation of the donor) c = Correction factor of acceptor only measurement

= Acceptor emission (by excitation of the donor) / acceptor emission (by excitation of the acceptor)

3.8.4 Dynamic FRET measurement by fluorescence microscopy

HEK 293 cells were mounted on an Axiovert 200 inverted microscope (Zeiss, Jena, Germany) with an oil immersion objective (100×), a dual emission photometric system (TILL Photonics, Planegg, Germany), and Polychrom V (TILL Photonics). Cells were illuminated at 435 nm (beam splitter DCLP 460, Chroma Technologies, Rockingham, USA) for ECFP excitation, or > 515 nm (beam splitter DCLP 505, Chroma Technologies) for EYFP excitation. To determine dynamic changes in

photobleaching. ECFP and EYFP emissions were recorded continuously, and FRET was determined as the ratio of EYFP / ECFP. Absolute FRET between ECFP and EYFP was determined by measuring donor quenching after acceptor photobleaching for 5 min by illumination at 500 nm. Fluorescence intensities were measured with a HEKA EPC-10 amplifier controlled by Patchmaster software (HEKA).