University of Heidelberg

Research Topics

 - Concepts for quantitative super-resolution imaging- dSTORM imaging with photoswitchable synthetic dyes

 - Coordinate-based colocalization analysis for single-molecule localization microscopy (SMLM)

 - Correlated high-throughput and high-resolution microscopy (in collaboration with H. Erfle, BioQuant Heidelberg)

 - Fluorogenic nucleic acid probes (in collaboration with A. Mokhir, University of Erlangen)

 - Photoswitching of synthetic dyes in phalloidin-dye-conjugates (in collaboration with T. Kuner, University of Heidelberg)

 - Structural organization of HIV proteins at the nanoscale (in cooperation with HG Kräusslich, University of Heidelberg)

Single-molecule localization microscopy

Fluorescence microscopy is a technique to visualize distributions of fluorescently labelled molecules within a sample in a non-invasive and specific way and is also compatible with complex biological systems like cells or tissues. However, the resolution is limited by the diffraction limit of light to about 200 nm in the lateral imaging plane and to >500 nm in the axial plane. In the past years, a variety of super-resolution techniques were developed circumventing the diffraction limit.

Single-molecule localization microscopy (SMLM) bypasses the lateral resolution limit by separation of fluorescence emission of fluorophores closer than the diffraction limit in time and their accurate localization. For this purpose, photoactivatable or photoswitchable fluorophores are used, thus enabling the reconstruction of high-resolution images. Typically, SMLM experiments are either performed in a wide-field configuration, or in total internal reflection (TIR). The advantage of TIR fluorescence microscopy is that the excitation of fluorophores is restricted to about 100 nm along the axial plane and thus reduces the observed axial depth. Two concepts that rely on photoswitching or photoactivation are direct stochastic optical reconstruction microscopy (dSTORM) and photoactivated localization microscopy (PALM).

As mentioned before, the key of SMLM is to optically modulate fluorescence emission and thus to limit the number of fluorophores detected at a given time to the single-molecule level. In the dSTORM approach, this is achieved by operating conventional synthetic fluorophores (i.e. commercially available, for example as antibody conjugates) as photoswitches in the presence of reducing buffers. dSTORM exploits the fact that most fluorophores are prone to reduction through suitable reagents that have a matching redox potential, and by that transit into a long-lived, non-fluorescent radical or other reduced state. The transition into this “dark” state is governed by the excitation light, the reducing potential of the buffer reagents (typically thiols such as mercaptoethylamine) and the intensity of the excitation light. The back-transition into the “bright” state is governed by residual oxygen, the thermal stability of the off-state and the intensity of a second wavelength (typically blue-shifted to about 100 nm to the excitation light). The dSTORM concept allows reversible photoswitching of many synthetic fluorophores; prominent examples are Alexa Fluor® 647, Alexa Fluor® 532, ATTO 655 and many others.

Different to dSTORM, the PALM concept was originally conceived with intrinsically photomodulatable fluorescent proteins, without the need of chemical buffers (and thus suited well for live cell imaging). PALM has the advantage that stoichiometric labeling through co-expression of fluorescent proteins is accessible. The drawback is their lower quantum yield compared to organic dyes, which leads to a lower localization accuracy. Common fluorescent proteins for PALM imaging are mEos, PAmCherry, Dronpa and others.

Labeling strategies

There is a huge toolbox available to specifically label proteins, nucleic acids, lipids as well as carbohydrates. The following section summarizes some of the labeling strategies used in the group.

Immunofluorescence

A standard procedure is immunolabeling using antibodies against the target molecule. Direct immunofluorescence uses a fluorophore labeled primary antibody directed against the molecule of interest. In the case of indirect immunofluorescence, a primary antibody binds to the target molecule and is detected by a secondary antibody labeled with an organic dye.

Conjugation to functional groups

Proteins can be directly coupled to an organic dye at specific amino acids, e.g. at amine groups (N-terminal amine or ε-amino group of lysine) via N-hydroxysuccinimide (NHS) ester coupling or at the thiol group of cysteine with maleimide-functionalized dyes.

Fusion proteins

Proteins also can directly be linked to a fluorescent protein. In this case the gene sequence of a fluorescent protein is fused to the gene of interest. The fusion protein is then expressed within the cell and live cell imaging is possible.

Chemical tags

Chemical tags like the SNAP- and Halo-tag use a marker protein fused to the protein of interest which then binds a fluorescent tag possessing a reactive group (e.g. O6-benzylguanine derivatives or chloroalkane conjugates).

DNA stains

Established DNA labeling procedures use intercalators, e.g. ethidium bromide which incorporates between base pairs of the DNA, or minor groove binders, e.g. DAPI. Click chemistry offers a powerful alternative for labelling nucleic acids. Here, a modified nucleoside exhibiting an alkyne group (e.g. ethinidyl-dU, EdU) is added to the growth medium of proliferating cells, and the nucleoside is incorporated in newly synthesized DNA. Via a copper(I)-catalyzed reaction an organic dye functionalized with an azide group is coupled to the modified nucleoside, and allows high-density labelling of DNA with virtually any fluorophore.

Contact: E-Mail (Last update: 01/10/2015)