BIOQUANT

Quantitative Microscopy

Background

Fluorescence microscopy is a well-suited technique for examining fixed and living specimens and both topological features as well as fluorescence intensity variations can be evaluated in a quantitative manner. Fluorescent compounds emit light in a defined spectral range when excited with light of a specific wavelength. Fluorescence imaging makes use of this phenomenon and allows for the selective and specific detection of molecules at small concentrations with a good signal-to-noise ratio. Furthermore, detection of the signal by confocal microscopy allows to acquire images with good resolution in the focal plane, suppressing any signal arising from out-of-focus regions.

Work of the group

In our lab, we use different fluorescence microscopy imaging techniques, e.g. wide-field, confocal laser scanning microscopy and automated 3D screening. In combination with image analysis, we detect and quantify the properties of different molecules such as DNA, RNA or proteins at specific cellular locations, which aids to deduce their functions and underlying molecular mechanisms.

Figure 1. Automated confocal microscopy for screening. 1. Implementation of screening 2. Automatic image analysis

 

Apart from imaging, fluorescence microscopy-based techniques are essential tools for measuring the dynamics of molecules in living cells to approach a variety of biological questions. The main fluorescence fluctuation microscopy techniques applied in our lab are fluorescence correlation spectroscopy (FCS) and fluorescence bleaching techniques (e.g. FRAP). A combination of these complementary methods allows for a comprehensive analysis on multiple length and time scales.

 

Figure 2. Fluorescence microscopy-based techniques. Left: Fluorescence Correlation Spectroscopy (FCS) is based on the diffusion of a small number of fluorescent particles through the focal volume. Analysis of the fluctuating signal allows to determine molecular properties, e.g. diffusion parameters or concentrations.Right: In Fluorescence Recovery After Photobleaching (FRAP) a defined region of the cell is bleached. The dynamics of the recovering intensity in this region are informative about the underlying process of diffusion and binding and unbinding of labeled molecules.

 

Selected references

 

Rademacher A, Erdel F, Trojanowski J, Schumacher S & Rippe K (2017). Real-time observation of light-controlled transcription in living cells. J Cell Sci published online 9 November 2017. doi: 10.1242/jcs.205534 | Abstract | Reprint (12.3 MB) | Article metrics.

Gunkel M, Chung I, Wörz S, Deeg KI, Simon R, Sauter G, Jones DT, Korshunov A, Rohr K, Erfle H & Rippe K (2017). Quantification of telomere features in tumor tissue sections by an automated 3D imaging-based workflow. Methods 114, 60-73. doi: 10.1016/j.ymeth.2016.09.014 | Abstract | Reprint (7.3 MB) | Article metrics.

Wachsmuth M, Knoch TA & Rippe K (2016). Dynamic properties of independent chromatin domains measured by correlation spectroscopy in living cells. Epigenetics Chromatin 9, 57. doi: 10.1186/s13072-016-0093-1 | Abstract | Reprint (5.1 MB) | Article metrics.

Osterwald S, Deeg KI, Chung I, Parisotto D, Wörz S, Rohr K, Erfle H & Rippe K (2015). PML induces compaction, partial TRF2 depletion and DNA damage signaling at telomeres and promotes alternative lengthening of telomeres. J Cell Sci 128, 1887-1900. doi: 10.1242/jcs.148296 | Abstract | Reprint (4.3 MB) | Article metrics.

Baum M, Wachsmuth M, Erdel F & Rippe K (2014). Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells. Nat. Commun. 5, 4494. doi: 10.1038/ncomms5494 | Abstract | Reprint (6.4 MB) | Article metrics.

Erdel F & Rippe K (2012). Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis. Proc. Natl. Acad. Sci. USA 109, E3221-E3230. doi: 10.1073/pnas.1209579109 | Abstract | Reprint (3.4 MB) | Article metrics.

Erdel, F. & Rippe, K. (2011). Binding kinetics of human ISWI chromatin-remodelers to DNA repair sites elucidate their target location mechanism, Nucleus 2, 105-112. doi: 10.4161/nucl.2.2.15209 | Abstract | Reprint.

Erdel F, Müller-Ott K, Baum M, Wachsmuth M, Rippe K. (2011). Dissecting chromatin interactions in living cells from protein mobility maps. Chromosome Res. 19(1):99-115.

 

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