Multifocal Fluorescence Fluctuation Microscopy
Our self-developed Spatial and Temporal Fluctuation Microscope (STFM) extends the point-confocal concept to multifocal illumination and detection along a line. It provides fast confocal imaging, allows for single particle tracking and high spatially resolved parallel detection of particle concentration fluctuations in Fluorescence Correlation Spectroscopy (FCS) experiments at hundreds of points. In addition to autocorrelation analysis, STFM provides a possibility for calculation of spatial cross-correlation curves between the fluorescence signals from different foci in the sample.
Figure 1: A, B Concept of data acquisition with the STFM. A The fluorescence originating from the line-shaped illumination volume is projected on a pixel line array of an EM-CCD camera. A fluorescently labeled particle in the illuminated volume gives rise to a Gaussian-shaped intensity profile on the line detector, which is described by the point-spread function. B The fluorescently labeled particle moves in, through, and out of the illuminated and detected line. This line is divided into several detection volumescorresponding to each pixel along the detector array. The particle movements will be detected as a series of short, randomized fluorescence signals at the corresponding pixel of the line detector.
The STFM system consists of a custom-designed line scanning laser illumination and fluorescence detection setup that is attached to an inverted Leica IRBE microscope. For fluorescence excitation, a Coherent Sapphire DPSS laser emitting at 488 nm is used. The line profile for illumination is generated with cylindrical lenses in the illumination beam path in such a way that in the intermediate image plane the laser light is focused to the diffraction limit in one direction and collimated in the other direction. A single-axis scan mirror adjusts the vertical line position, whereas horizontal scanning is not necessary due to the line profile. Fluorescence from the illuminated line is imaged on a single line of an electron-multiplying charge-coupled device (EM-CCD). A confocal slit is positioned in a conjugate image plane in order to provide optical sectioning. The spatial resolution of 200 nm in lateral direction perpendicular to the line, 380 nm along the line and 700 nm in axial direction was measured with quantum dots. The temporal resolution of 14 Ás is limited by the readout time of the EM-CCD camera.
The Spatial and Temporal Fluctuation Microscope allows for the application of bleaching techniques such as Fluorescence Recovery After Photobleaching (FRAP) or Continuous Photobleaching (CP), which will be implemented. Moreover, particle tracking concepts that allow for distinguishing directed from diffusive motion will be tested with the STFM.
Heuvelman G. (2008).
Development and design of a spatially and temporally resolved fluorescence fluctuation microscope for the analysis of molecular mobilities and interactions.
PhD Thesis, Ruprecht Karl University of Heidelberg.
Heuvelman G, Erdel F, Wachsmuth M, Rippe K. (2009).
Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy.
Eur Biophys J. 38(6):813-28.
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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