Minimal-Invasive Fluorescence Microscopy

Ultra-low excitation light (nW) and ultra-low label concentration (1ethidium bromide in 10.000 DNA base-pairs) [1] is required to preserve the living state in fluorescence microscopic cell studies. Photodynamic reactions can occur even at very low excitation levels [2]. DASPMI-labelled mitochondria may serve as an example. Ultra-sensitive imaging detectors (QA-MCP-PMT) are required that have time-resolution < 10 ps at a dynamic range of > 106, or even > 107 with cooled photocathode and “hot” MCP-technology.

[1] M. Tramier, K. Kemnitz, C. Durieux, J. Coppey, P. Denjean, R.B. Pansu, and M. Coppey-Moisan, Restrained Torsional Dynamics of Nuclear DNA in Living Proliferative Mammalian Cells, submitted 1999. [2] Maite Coppey-Moisan et al.

-Nature is Time-Correlated-

Static intensity technology (CCD) is blind towards the huge pool of information, residing in the environment-sensitive fluorescence lifetime of a molecular probe. In addition, time-correlation of the emitted fluorescence leads to dramatic reduction of background, which is non-correlated in respect to the pulsed laser excitation. Application of the unique time-correlated single photon counting (TCSPC) technique [3], and its recent variant of time-and space-correlated single photon counting (TSCSPC) [4] in cell biology fluorescence studies, increases sensitivity by 10-100 times compared to steady-state CCD technology.

[3] D.V. O‘Connor and D. Phillips, (1984) Time-Correlated Single Photon Counting, Academic Press, New York. [4] K. Kemnitz et al.