Link to Core Unit Fluorescence Imaging
A powerful technology platform is available in the field of imaging at the RVZ:
Dr. Katrin Heinze
Tel.: +49 (0)931 31-84214
Mike Friedrich (Microscopy and Optical Engineering)
The following techniques are available to all research groups:
Digital Time-Lapse Microscopy
Dynamic cellular processes, such as cell migration and invasion, cytokinesis, and cell survival/apoptosis can be monitored by time-lapse microscopy (bright field and fluorescence mode) at 37°C. Controlled experiments monitoring 2D or 3D cell cultures can be performed for up to 7 days. Time-lapse images can be assembled into a movie and analyzed by specialized software packages. Thus, processes such as cell migration can be followed and quantified by automated computer-assisted cell tracking.
Confocal Fluorescence Microscopy
Confocal fluorescence microscopy has become one of the most powerful and versatile tools in the life sciences. With confocal Laser Scanning Microscopy (LSM), it is possible to exclusively image a thin optical slice within a thick specimen (up to 100 µm thick), a technique known as 'optical sectioning'. Thus, the thickness (z-dimension) of such an optical slice is typically less than 1 µm.
Microscope: Leica SP5 (with visible (405, 458, 476, 488, 496, 514, 561, 594, 633 nm) and IR-laser sources)
The laser scanning microscope (LSM) or confocal microscope offers a large variety of imaging features and analysis methods. The most important and frequently used techniques are listed below:
Fluorescence Resonance Energy Transfer (FRET)
FRET is a method to investigate protein interactions. Interacting proteins or molecular complexes are labeled with two spectrally distinct fluorophores, one serving as the ‘donor’ transferring the excitation energy to the other called the ‘acceptor’. This energy transfer largely depends on the distance between them, which means FRET can be used as a very sensitive molecular ruler to probe conformational changes of a protein complex or/and intramolecular interactions. Read out is the fluorescence of donor and acceptor molecules.
Microscope: Leica SP5
Fluoreszenz Lifetime Imaging (FLIM)
One key-indicator for FRET (above) is the fluorescence lifetime of the interacting fluorophores which changes upon energy transfer. The fluorescence lifetime is like a ‘fingerprint’ of the molecule in its specific environment and defines the characteristic delay time between the (instantaneous) absorption of a photon and the emission of a fluorescence photon. With the FLIM-technique we can determine the fluorescence lifetime of individual biomolecules in a confocal fluorescence image, pixel by pixel, to exactly quantify FRET (one- and two-photon excitation).
Microscope: Leica SP5
Fluorescence Recovery After Photobleaching (FRAP)
FRAP is a technique to unravel dynamic processes in (living) cells. For FRAP, fluorescently labeled proteins are illuminated with scanned focused laser light until they photobleach. If fluorescence returns to the previously bleached area, this indicates molecular dynamics, since the proteins carrying the bleached fluorophore must have been replaced by surrounding proteins with non-bleached fluorophores. Evaluating the time constants of this fluorescence recovery can quantify the underlying dynamics, e.g. to calculate diffusion and transport constants. (Minimal temporal resolution: 100 ms, bleach area: 4 x 4 µm or "single-spot").
Microscope: Leica SP5
Fluoreszenz Korrelations Spektroskopie (FCS)
FCS is a statistical single-molecule technique to determine sub-micron diffusion and transport constants well as photophysical properties. FCS measures fluorescence fluctuations of fluorescently labeled molecules when they enter or leave the focal spot. These fluorescence fluctuations contain valuable information about the dynamic constants of the moving molecules that can be quantified by autocorrelation of the fluctuations and applying appropriate fitting models. Very sensitive detectors (single photon counters) are required to perform FCS (home-built with Pico Quant detection).
Further Microscope: Leica SP5.
Light Sheet Fluorescence Microscopy (LSFM)
Light Sheet Fluorescence Microscopy (LSFM), often called 'Single Plane Illumination Microscopy' or 'Selective Plane Illumination Microscopy' (SPIM), is a relatively new technique that allows for 3D imaging of large tissue specimens or whole organs with cellular resolution. Only a single plane of the sample will be illuminated at a time. Before imaging the sample has to be made transparent by dehydration. Water is replaced by an oil-like substance with the same refractive index as protein. After preparing the sample the organ appears glassy, highly transparent and scatters less illumination light.
Microscope: Zeiss Axiovert 200, Reference: Huisken, J., et al. (2004) 305, 1007 or Dodt, H.U., et al., N. Methods , (4) 331.
Two-Photon Laser Scanning Microscopy
Two-photon microscopy is a powerful technique to study delicate tissue and cells in vivo and in vitro. Particularly intravital studies benefit from the enhanced penetration depth of up to 0.5 - 1 mm and improved depth of focus, both due to excitation by infrared laser light at wavelengths where biological samples appear largely transparent and scatter less light (compared to visible light). Moreover, phototoxic effects are reduced since the photo-damage outside the focus region is negligible.
We offer two different two-photon systems
- based on an inverse microscope stage (Leica SP5), and thus well suited for cell culture and tissue studies (ex vivo, in vitro).
- based on a fixed stage (LaVision Trimscope), and thus well suited for intravital microscopy.
Both Systems offer a continuously tunable wavelength-range for fluorescence excitation of 700-980 nm (Ti:Sa laser) und 1100-1400 nm (OPO). Second harmonic generation (SHG) based imaging is also possible and often useful for microscopy of highly ordered noncentrosymmetric molecular structures such as collagen and certain muscle tissue.
A STED microscope (STED: STimulated Emission Depletion) is particularly suitable for imaging live and fixed cells. Based on a confocal microscope, the spatial resolution of a STED microscope is typically 3 times better than a confocal system due to the STED beam: An additional pulsed laser with a special excitation profile allows for a spatial resolution of up to 80 nm in cells. The temporal resolution is comparable to a confocal microscope, and dependent on the scan speed.
Note, only certain dyes are suitable for STED microscopy (e.g. Atto647).
Our STED microscope has the following features:
1. Inverted stage
2. Single color STED microscopy, combinable with up to 3 other confocal color
3. Pulsed STED-laser, und up to 4 cw-laser wavelengths (458-633 nm, no
Single Molecule Imaging by Atomic Force Microscopy
Contact: Group Tessmer
Atomic force microscopy (AFM) uses a fine probe to mechanically scan biological samples, producing topographical images with resolutions in the order of a few nanometers. This allows us to visualize individual biological molecules such as proteins and their interactions. The high resolution of AFM imaging combined with its applicability in liquid physiological environments positions this technique as a complementary link between X-ray crystallography (with atomic resolution in the order of Ångstroms to obtain structural information about molecules) and functional in vitro and in vivo analyses of biological systems using optical microscopy approaches (with typical resolutions of a few hundred nanometers).
Microscope system: Asylum Research MFP-3D
Total Internal Reflection Microscopy (TIRFM)
Contact: Gohla Group
TIRF microscopy allows studying dynamic processes in live cells in or near the plasma-membrane. TIRFM creates an extremely narrow fluorescence excitation field in the sample so that only fluorophores between 70 to 350 nm above the coverglass are excited. This inherent optical sectioning improves the z-axis specific resolution. For biological cells plated on the coverglass, only the basolateral membrane surface within the excitation depth will be excited, and thus only this region will be observed with fluorescence microscopy. With the available microscope, dynamic processes can also be analyzed by FRET experiments within the TIRF excitation field to investigate protein dynamics in plasma membranes.
Microscope: Leica AM TIRF Microscope (dual color detection with visible laser sources)