AVS 50th International Symposium
    AVS 50th Anniversary Plenary Session Tuesday Sessions
       Session AP-TuM

Invited Paper AP-TuM1
Light Optical Nanoscopy in Cellular Biophysics

Tuesday, November 4, 2003, 8:20 am, Room 310

Session: Information
Presenter: C. Cremer, Univ. of Heidelberg, Germany
Authors: C. Cremer, Univ. of Heidelberg, Germany
U. Spoeri, Univ. of Heidelberg, Germany
A.V. Failla, Univ. of Heidelberg, Germany
B. Albrecht, Univ. of Heidelberg, Germany
Ch. Wagner, Univ. of Heidelberg, Germany
A. Schweitzer, Univ. of Heidelberg, Germany
L. Hildenbrand, Univ. of Heidelberg, Germany
I. Upmann, Univ. of Heidelberg, Germany
J. Rauch, Univ. of Heidelberg, Germany
G. Kreth, Univ. of Heidelberg, Germany
N. Kepper, Univ. of Heidelberg, Germany
Ch. Engelbrecht, Univ. of Heidelberg, Germany
A. Rapp, Inst. for Molecular Biotech., Germany
M. Hausmann, Univ. of Freiburg, Germany
D. Toomre, Yale Univ.
S. Martin, Medical Res. Council, UK
A. Pombo, Medical Res. Council, UK
T. Cremer, Univ. of Munich, Germany
Correspondent: Click to Email
For many studies in cellular biophysics, it is highly desirable to develop optical methods for the analysis of specific biological nanostructures in the interior of three-dimensionally conserved cells. Here, important structural parameters to be considered are topology, i.e. mutual positions and distances of constituting subunits, as well as information about the size of such objects. In the low energy range, this has become possible by development of novel methods of far-field light fluorescence microscopy. Spectral Precision Distance Microscopy [SPDM] is based on labelling of neighbouring objects with different spectral signatures, spectrally selective registration, high precision position monitoring, and careful calibration of chromatic aberrations, cross talk etc. In combination with confocal laser scanning microscopy, SPDM allowed the measurement of spatial positions and mutual distances ("topology") of DNA sequences in specific human nuclear gene domains down to the 30 - 50-nanometer range. Theoretical considerations supported by "Virtual Microscopy" computer simulations indicated that using "Point Spread Function (PSF) Engineering" approaches with a suitably modified PSF, even at the fluorescence photon count number typical for single molecule fluorescence emission, a topological resolution limit down to the few-nanometer range with a precision in the subnanometer range might become feasible. For example, Spatially Modulated Illumination [SMI] far field light microscopy provides a PSF with the required properties; presently, experimental distance measurements in the direction of the optical axis down to the few nanometer scale, with a precision in the one-nanometer range (about 1/500 of the exciting wavelength) have been realized. Furthermore, SMI-approaches have been used to measure the diameter of individual fluorescent targets down to a few tens of nanometer, corresponding to about 1/16 of the exciting wavelength used.