crc press - E-Lib FK UWKS

Model Amphipathic Peptides 89
At the end of each protocol the integrity of the plasma membrane was verified
by addition of trypan blue and measurement of the trypan blue fluorescence (λexc =
543 nm, λem > 590 nm).
To monitor the fluorescence images and measure the fluorescence intensities an
LSM 410 invert confocal laser scanning microscope (Carl Zeiss, Jena GmbH, Jena,
Germany) was used. Excitation was performed at 488 nm (Fluos) or 543 nm (Trypan
Blue) using an argon-krypton or helium-neon laser and a dichroitic mirror FT 510
or NT 543/80/20 for respective wavelength selection. Emission was measured at
wavelengths >515 or >570 nm with cut-off filters LP 515 and LP 570, respectively,
in front of the detector. The fluorescence intensities inside and outside the cell were
recorded in predetermined regions of interest (ROIs, 16 × 16 pixel; 30 scans with
a scan time of 2 sec with double averaging) as described by Lorenz et al. 30 The
background fluorescence intensity was determined before addition of the peptide
solution for 300 sec. For measurement of the intracellular fluorescence, three ROIs
were selected in the cytosol and one in the nucleus of three cells in such a manner
that the diffuse fluorescence could be recorded without interference with vesicular
fluorescence.
The intracellular fluorescence signal was corrected for contributions from the
extracellular fluorescence arising from nonideal confocal properties of the CLSM
by estimating the distribution function of sensitivity in the Z direction of the microscope,
according to Wiesner et al. 31
4.6.4.2 Measurement of Axial Response of the Confocal System
Dye solution of 400 µl (carboxyfluorescein or fluorescein-labeled peptide; 1.25 µ M)
was transferred to the cover slip. The fluorescence of the dye and the reflection
signal of the excitation source were detected in a two-channel measurement. The
scan plane (XY-plane) was moved in 1-µm steps in the Z direction up to 200 µm,
beginning at the middle of the cover slip. Adjacent to the optical boundary (cover
slip-dye) the step width was decreased to 0.2 µm. At each step a two-channel image
(512 × 512 pixel) with a scan time of 2 sec and a double averaging was captured.
The intensities of the fluorescence and reflection signals were obtained directly from
the single-channel image after each scan. The values of the intensities were stored
according to the Z position. The registration of the reflection signal was used for
determining the Z position of the optical boundary.
4.6.4.3 Calculation of Contributions from Out-of-Focus Planes
to Intracellular Fluorescence Intensity
After preincubating the cells for 10 min with 1.25 µM solutions of the dye or the
flurescently labeled peptide respectively, and subsequent transfer of 400 µl of the
cell suspension to the cover slip, optical measurements were performed using the
experimental setup that follows.
The XY-plane was positioned in the middle of a cell in the Z-axis using the
reflection and the transmission modes of the microscope. Regions of interest (ROIs,
16 × 16 pixel) were fixed between the center of the cell and the extracellular medium.