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Microscopy
Confocal laser scanning microscopyConfocal microscopy differs from conventional light microscopy in the light source, the detection, generation and resolution of acquired images. In a conventional fluorescence microscope the whole specimen is illuminated by a certain excitation wavelength, likewise emitted light is gathered from all planes of the specimen. Visible light cannot be focused in a single plane, therefore not only emitted light from the focal plane but below and above the specimen are detected. This additional light detected results in a blurry image of decreased contrast and resolution, especially for thick specimens. In contrast to conventional light microscopy, in confocal microscopy a defined spot in the focal plane of the specimen is illuminated at a certain time point. Laser light sources of defined excitation wavelengths are used. The laser light is focused by passing through a very small aperture, such as a pinhole or a slit. Furthermore, emitted light from below or above the focal plane is eliminated by preventing passing a second pinhole (see figure 3.6, orange lines). By changing the z-axis distance between specimen and objective lense, the focal plane can be adjusted and so called optical slices of the specimen in X-Y plane can be generated. This technique is a non-invasive approach to investigate both fixed and living cells. Emitted light of an illuminated spot in the focal plane is detected by photomultiplier tubes (PMT), which enhance signal. Series of illuminated PMT outputs are processed to an image.
Fluorescence Microscopy of Fixed CellsSolutions:
293 or HeLa cells are passaged and seeded at a density of 500.000 cells/6-well (40 % confluence), each 6-well contains a round sterile glass cover slip. 1 day after lipofectamine or CaCl2 transfection of 293 or HeLa cells with fluorescent fusion proteins, the culture medium is removed, the cells are washed once with PBS def. and about 2 mL 4 % paraformaldehyde solution per 6-well are carefully pipetted to the cells. After about 10 minutes incubation at room temperature the paraformaldehyde solution is removed. The fixed cells are washed with PBS and covered with mounting medium. Then the coverslip is carefully taken out of the 6-well and placed on a glass slide with the cells facing the slide, trying to avoid bubbles. Excess of mounting medium is removed and clear nailpolish is applied on the edges of the coverslip to seal the fixed sample to prevent drying. Fluorescent microscopy is then performed with the sample.
Live Cell Fluorescence Microscopy
293 or HeLa cells are seeded in 6-well plates containing round sterile coverslips (diameter: 15 mm), the next day lipofectamine or CaCl2 transfection is carried out. 1 day after transfection the round coverslips are carefully taken out of the well and pressed on the hole of an aluminum slide (figure 1.7). The edges of the aluminum slides are greased to seal the cover slips. About 70 µL of DMEM complete medium is pipetted on the cells on the coverslip. With a second blank coverslip pressed on the other side of the aluminum slide a chamber filled with medium is created. The aluminum slide is placed on the object table with the cells grown on the coverslip on the bottom.
Fluorescence Recovery after Photobleaching (FRAP) on Zeiss LSM510
Halftime for recovery (diffusion): t1/2 = 0.69 / k
Fluorescence resonance energy transfer (FRET) microscopyHeLa, 293 or endothelial cells were seeded on 23 mm round glass coverslips in 6-well plates (4x 105 cells per well) and transfected with ECFP-IKK2 and EYFP-IKIP. One day after transfection, coverslips were mounted on a self-made perfusion chamber and living cells were imaged by fluorescence microscopy using a Nikon Diaphot inverted microscope equipped with a cooled CCD-camera (Kappa, Bad Gleichen, Germany) and filter sets that discriminate between ECFP and EYFP fluorescence (Omega Optical Inc., VT, USA). FRET microscopy was carried out by detecting the increase in donor (ECFP) fluorescence after photodestruction of the acceptor (donor recovery after acceptor photobleaching). For that purpose, cells were imaged with an oil immersion objective and images were captured with the donor filter using a 90% neutral density filter to prevent donor bleaching. This was followed by bleaching of the FRET acceptor (EYFP) with the appropriate filter set in the absence of the neutral density filter using a 100W Mercury lamp for about 45 – 60 sec. Subsequently, the neutral filter was again included into the excitation light path and another image was taken with the ECFP-filter set under the same camera setting as the first one. An increase in the donor-fluorescence intensity was visualized by calculating a ratio image of the ECFP-image before and after acceptor photobleaching using NIH-Image software or the Windows™-equivalent ScionImage (Scion Corporation Inc. Maryland, USA). As alternative to the donor recovery technique, the 3-filter method of FRET microscopy was applied as described (Youvan et al., 1997: Youvan DC, Coleman WJ, Silva CM, et al.: Calibration of fluorescence resonance energy transfer in microscopy using genetically engineered GFP derivatives on nickel chelating beads. Biotechnology et alia 1997, 3:1--18. (= online journal: http://www.et-al.com/); FRET techniques are also reviewed in Schmid & Sitte, 2003: Schmid JA, Sitte HH. Fluorescence resonance energy transfer in the study of cancer pathways.Curr Opin Oncol. 2003 Jan;15(1):55-64.)
In Situ Nick Translation (ISNT) to detect apoptotic cells1. Make cryo sections of human skin (7 µm thick) and mount them on poly-L-lysine coated cover glasses. Let the sections dry for 30 min at room temperature. 2. Fixation in acetone (precooled) for 10 min at 4°C. 3. Wash in PBS for 5 min at r.t. 4. ISNT-reaction: 100 µl/ cover glass; 40 min at r.t.: amount for 1 ml reaction mixture 3 µM FITC-12-dUTP (Boehringer 1373242): 3.75 µl (0.8 mM stock solution) (or 3 µM Biotin-16-dUTP) 3 µM dGTP 7.5 µl (0.4 mM stock) 3 µM dATP 7.5 µl (0.4 mM stock) 3 µM dCTP 7.5 µl (0.4 mM stock) DNA-Polymerase I (Boehringer 642711) 50 units/ml (10 µl of 5 u/µl stock) (endonuclease-free) 10x reaction buffer (50 mM Tris/HCl pH 100 µl 10 mM MgCl2 0.1 mM DTT) A. dest nuclease-free ad 1 ml 5. Washing with PBS: 3 times for 5 min at r.t. 6. Protein block: 30 min at r.t. with 10% FCS in PBS 7. Incubation with peroxidase-conjugated anti-FITC (Boehringer; 1:25 in block solution; 30 min at 37°C) 8. Washing with PBS: 3 x 5 min at r.t. 9. Metal-enhanced DAB-staining (Pierce, 34065): 100 µl/cover glass: Incubation about 5 - 20 min (r.t.) Mounting and conventional microscopy (maybe after hemalaun counterstaining)
Alternative: (if Biotin-16-dUTP was used): 7. Incubation with FITC- or Texas Red conjugated streptavidin (Amersham) 8. Washing with PBS: 3 or 4 times for 5 min at r.t. 9. Mounting and fluorescence or laser scanning microscopy
Immunofluorescence StainingsGem stainFixation: 5 min with Methanol (-20°C)
IkB stainFixation: 15 min 4% Paraformaldehyd
p65 (RelA)
NIK
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