Microscopy

Confocal laser scanning microscopy

Confocal 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 Cells

Solutions:

  • 4 % (w/v) paraformaldehyde pH 7.4 in PBS

  • mounting medium: glycerol in PBS (1:6)

  • round glass cover slips (Ć 15 mm) , sterile, stored in 70 % ethanol

 

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

  • greased aluminum slide

 

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

  1. Capture an image of the whole cell before bleaching

  2. Define a bleaching / scan region (and maybe in addition another scan region that is not bleached)

  3. Perform a time series with 1 scan prebleach, about 70 iterations of bleaching with 100% laser power and then 50-100 scans of the bleach region (and also the non-bleached control region if you specified one)- a good time resolution can only be obtained if just the small bleach region (and maybe the control region) is scanned - and not the whole cell; averaging of 2 or 4 scans reduces the electronic noise and leads to better quantifications.

  4. Capture an image of the whole cell after the FRAP time series (with the same conditions as the prebleach image – for calculating the total loss of fluorescence.

  5. Using LSM Image Browser (Freeware from Zeiss) export the images and the time series in TIF format (in a new folder for each time series) and record the seconds of the single images (seconds: see values in the slices-view: type first 2 of the series in the Excel sheet and extend the column to the final value)

  6. Using NIH-Image (ScionImage for Windows) open the TIF files: measure the mean fluorescence in a control region or for the whole cell for both the prebleach and the postbleach images and calculate the loss of overall fluorescence due to the bleaching in the region of interest.

  7. Open all the TIF images of the FRAP time series (using the “open all” command)

  8. Measure the bleach/scan-region of all images using the  "measure all" command of the Measure Macro (white is zero, black is 255)
    (measure also the mean fluorescence of the control region, if you recorded that)

  9. Copy fluorescence raw data from the results window to the corresponding column in the Excel sheet

  10. Calculate the difference of mean fluorescence from the background and normalize the fluorescence values to 100% for the initial fluorescence.

  11. Divide the percent values by the correction factor calculated from the total loss of fluorescence (e.g. if total fluorescence decreased from 1 to 0.9 then divide the mean fluorescence of the FRAP regions for each time value by 0.9 to compensate for the loss in total fluorescence). A similar compensation can be obtained by normalizing the FRAP fluorescence values to the control scan region that was not bleached. This method also compensates more exactly for the bleaching effect in the course of scanning of the time series (this scanning-dependent bleaching effect is opposed to the recovery of fluorescence in the bleach region due to diffusion of non-bleached molecules in the bleach region). This “dynamic correction” gives a somewhat better estimation of the curve (and the kinetics of the recovery) – but leads in principle to results that are very similar to the curve obtained with the “constant correction factor” (by calculating the total loss in fluorescence based on the intensities of the images that were captured before and after the FRAP-time series)

  12. For non-linear regression analysis (curve fit of the data to a single exponential association algorithm): Copy the data to a fitting program (such as GraphPad Prism) and perform the fitting with a “bottom to span” algorithm:

 

 

Halftime for recovery (diffusion): t1/2 = 0.69 / k

 

 

Fluorescence resonance energy transfer (FRET) microscopy

HeLa, 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 cells

1.  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 Stainings

Gem stain

Fixation: 5 min with Methanol (-20°C)

  1. 3x 5 min mit TBST wash (50mM Tris-HCl pH7.4, 150 mM NaCl, 0.1%Triton)

  2. Block: 1 h at RT with 3% BSA in TBS

  3. Incubation with 1. AK: anti-Gem (rabbit polyclonal, sc-371 Santa Cruz)
    1:250 (maybe better: 1:100) in TBS/3% BSA,  over night at 4°C (or 1 h at 37°C).

  4. 2x 5 min wash with TBST, 1x 5 min with TBS

  5. Incubation with Alexa488 (or FITC or similar) anti-goat 1:2000 in TBS/BSA: 1 h at 37°C

  6. 3x 5 min wash with TBST, 1x 5 min with TBS

  7. Mounting in PBS/Glycerol (1:7) – sealing with nail polish
    (or mounting with Mowiol or Dako Fluorescent Mounting Fluid)

 

IkB stain

Fixation: 15 min 4% Paraformaldehyd

  1. 3x 5 min mit TBST wash (50mM Tris-HCl pH7.4, 150 mM NaCl, 0.1%Triton)

  2. Block: 1 h at RT with 3% BSA in TBS

  3. Incubation with 1. AK: anti-IkB (rabbit polyclonal, sc-371 Santa Cruz)
    1:300 in TBS/3% BSA,  over night at 4°C (or 1 h at 37°C).

  4. 2x 5 min wash with TBST, 1x 5 min with TBS

  5. Incubation with Alexa488 goat anti-rabbit 1:2000 in TBS/BSA: 1 h at 37°C

  6. 3x 5 min wash with TBST, 1x 5 min with TBS

  7. Mounting

 

p65 (RelA)

  1. 3x 5 min mit TBST waschen (50mM Tris-HCl pH7.4, 150 mM NaCl, 0.1%Triton)

  2. Block: 1 h bei RT mit 3% BSA in TBS

  3. Inkubation mit 1. AK: anti-p65 NF-kB (rabbit polyclonal, sc-109, Santa Cruz)
    1:200 in TBS/3% BSA,  über Nacht bei 4°C (oder 1 h bei 37°C).

  4. 2x 5 min mit TBST waschen, 1x 5 min mit TBS

  5. Inkubation Alexa 488 goat anti-rabbit 1:2000 in TBS/BSA: 1 h bei 37°C

  6. 3x 5 min mit TBST waschen, 1x 5 min mit TBS

  7. Mounting

 

NIK

  1. 3x 5 min mit TBST waschen (50mM Tris-HCl pH7.4, 150 mM NaCl, 0.1%Triton)

  2. Block: 1 h bei RT mit 3% BSA in TBS

  3. Inkubation mit 1. AK: anti-NIK (rabbit polyclonal, sc-7211 Santa Cruz)
    1:100 in TBS/3% BSA,  über Nacht bei 4°C (oder 1 h bei 37°C).

  4. 2x 5 min mit TBST waschen, 1x 5 min mit TBS

  5. Inkubation Alexa 488 goat anti-rabbit 1:2000 in TBS/BSA: 1 h bei 37°C

  6. 3x 5 min mit TBST waschen, 1x 5 min mit TBS

  7. Mounting