Fluorescence microscopic methods and fluorescence staining

3.11.1 Epi-fluorescence microscopy and confocal laser scanning microscopy

3.11.1.1 Staining of the actin cytoskeleton

Fluorescence microscopy was used to examine the actin-cytoskeleton arrangement in MCDK-II cells, which were treated with drugs interfering with the actomyosin network or were cultured on porous substrates, and NMuMG cells before and after epithelial-to-mesenchymal transition. Staining of MDCK-II samples grown on porous substrates was performed by Helen Nöding in her master thesis. NMuMG cells were stained by David Schneider (Georg-August-University, Göttingen, Germany) To stain the actin cytoskeleton, cells were first rinsed 2 times using PBS and fixed using 4% PFA (FLUKA, Switzerland) for 15 minutes at room temperature. Afterwards, samples were rinsed 2 times with PBS and incubated for 45 minutes with blocking buffer (5% bovine serum albumin (BSA) and 0.3% Triton-X-100 in PBS) at room temperature. After removal of the blocking buffer, a 165 nM Alexa Fluor® 546 Phalloidin (Life Technologies, Carlsbad, CA, USA) in dilution buffer (1% BSA and 0.3% Triton-X-100 in PBS) was applied and samples were incubated for 1 hour. In a next step, samples were washed 2 times for 5 minutes using washing buffer (0.1% BSA in PBS). Finally, DNA was stained using a 500 ng/ml solution 4,6-diamidin-2-phenylindol (DAPI). After 10 minutes incubation, samples were again washed 2 times for 5 minutes using PBS.

The samples were examined using either an Olympus BX51 epi-fluorescence setup (Olympus, Tokyo, Japan) or a confocal laser scanning microscope (LSM 710, Zeiss, Göttingen, Germany) equipped with an Argon-laser (LASOS Lasertechnik, Jena, Germany).

3.11.1.2 Sample preparation for fluorescence microscopic examination of GUVs exposed to nanoparticles

To examine the effect of nanoparticles on artificial lipid bilayers, GUVs prepared by electroformation (see chapter 3.4) were added into a solution of nanoparticles in buffer prepared either in ibidi µslide 8 well (ibidi, Martinsried, Germany) and 96-well plates for

examination using epi-fluorescence (IX81, Olympus, Tokyo, Japan) or in glass bottom petri dishes (MatTek, Ashland, MA, USA) for studies using confocal laser scanning microscopy (LSM 710, Zeiss, Göttingen, Germany). Prior to experiments, glass bottom petri dishes were treated with a 0.1% BSA solution in deionized water for 1.5 hours to passivate hydrophilic glass surface and prevent GUVs from spreading.

3.11.1.3 Uptake of pyranine-stained nanoparticles solutions into A549 cells

To examine the uptake of nanoparticles, which are not fluorescently labeled themselves the plasma membrane of A549 cells was first stained using a 5 μg/ml Cell Mask™ Deep Red Plasma Membrane Stain (Life Technologies, Carlsbad, CA, USA) solution in cell culture medium at 37°C. After 5 minutes the staining solution was replaced by cell culture medium and incubated at 37°C and 5% CO2 for 15 minutes.

Then, the normal cell culture medium was replaced by 5 µg/ml nanoparticle solution in cell culture medium, which additionally contained 2 mM pyranine. The cells were incubated for 30 minutes at 37°C and 5% CO2. Afterwards, cells were rinsed using PBS and fixed using a 4% PFA solution in PBS. The samples were imaged using a confocal laser scanning microscope (LSM 710, Zeiss, Göttingen, Germany) equipped with an Argon-laser (LASOS Lasertechnik, Jena, Germany).

For data analysis, maximum intensity projections of the pyranine channel of z-stack images were subject to grain analysis using the open source software Gwyddion (http://gwyddion.net). The following parameters were used for grain detection: height = 20%, slope = 12% and curvature = 100%. Histograms of the equivalent radii of the found grains were created using IGOR Pro (WaveMetrics, Lake Oswego, OR, USA).

3.11.2 Fluorescence near metal surfaces

3.11.2.1 Experimental setup and data evaluation

A description of the experimental setup and data evaluation can be found in the publication of Chizhik, Rother et al.. (Chizhik et al., 2014a) In brief, a conventional home build confocal laser scanning microscopy setup, which is able to perform fluorescence lifetime measurements (see also chapter 4.4.2), was used with one modification: the cover slide was coated with a 20 nm gold layer. The acquired data were analyzed by Dr. Alexey Chizhik (III. Physical Institute, Georg-August-University,

Göttingen, Germany) according to the model of Chance, Prock and Salbey, which correlates the fluorescence lifetime of a fluorophore to its distance from a metal layer.(Chance et al., 1978) Due to energy transfer from the excited state of the fluorophore to the metal layer the fluorescence is quenched in a distance dependent manner. In general, the model requires the following input parameters: 1) the quantum yield of the fluorophore in free solution, 2) the emission spectrum of the fluorophore and 3) the dielectric function of the metal layer at the used wavelengths.

3.11.2.2 Sample preparation for metal-induced energy transfer fluorescence lifetime imaging and staining of the plasma membrane

For metal induced energy transfer- (MIET-) fluorescence lifetime imaging (FLIM) experiments, approximately 20,000 MDA-MB-231 cells were seeded into a small reservoir with a diameter of 1 cm, which was melted on top of the cover slide. Cell-substrate distance was monitored with MIET FLIM within two days after seeding. A549 cells and MDCKII were seeded in the same density on gold-coated glass bottom petri dishes (MatTek, Ashland, MA, USA).

Prior to the measurement, the plasma membrane of cells was stained by incubating living cells with HEPES-buffered cell culture medium containing 5 μg/ml Cell Mask™

Deep Red Plasma Membrane Stain (Life Technologies, Carlsbad, CA, USA) for 5 minutes at 37°C. For the actual measurement, the staining medium was replaced by HEPES- buffered cell medium, which allowed the investigation of living cells up to one hour after staining at 37°C. 
T o examine the spreading of MDCK-II cells, the cells were released from the culture flask by trypsinization for 5 minutes (trypsin/EDTA 0.5%/0.2%, Biochrome, Berlin, Germany) and kept in suspension in a density of 1×10⁶ cells/ml. The cells were used for up to 1 hour. For the adhesion experiments the gold-coated glass bottom petri dishes were incubated for 30 minutes with HEPES buffered cell medium. The cell medium was replaced by 400 μL of the staining solution (cell culture medium containing 5 μg/ml Cell Mask™ Deep Red Plasma Membrane Stain) and 50,000 cells were added. After 5 minutes at 37°C the staining solution was carefully replaced by HEPES-buffered cell culture medium and the measurement was started.