Parallel fluorescence spectroscopy tools for micro and nano-analytical applications down to single biomolecules


All biological processes rely on the complex interactions of biomolecules such as proteins, DNA, RNA, lipids and sugars. These interactions usually take place in large molecular assemblies. Typical examples are the “molecular machines” involved in signal transduction, transcription and translation of genomic information, protein degradation, or intracellular transport processes. A better understanding of these processes is paramount for a better understanding of the (patho)physiology of tissues and organisms and gives a base for gaining a better insight in key medical issues, such as the origin and growth mechanisms of tumors. Analytical techniques that provide information on these interactions are nowadays currently in use not only at the cutting edge of biomolecule research, but also in laboratories of medical and biological ambients. Fluorescence microscopy and spectroscopy is widely employed [1]. FRET (Förster resonance energy transfer in acceptor/donor pair) is employed for measuring intermolecular distances on a nanometer scale, thus obtaining information about the tagged proteins and their environment. Established FRET techniques rely on measurements of changes either of the spectral intensity or of the lifetime of the fluorescence.
FRET measurements of spectral intensity and of lifetime taken individually do not give a complete information about the involved fluorophore characteristics. There is a wide consensus among experimenters that for a better understanding of the biological process involved at cellular level it is necessary to acquire at every cell region under investigation simultaneously the spectral and temporal fluorescence data. This requires to develop optoelectronic instrumentation capable of carrying out spectrally resolved fluorescence- lifetime imaging microscopy (sFLIM). The only commercially available sFLIM detection system [2] employs as detector a multi- anode photomultiplier tube (PMT) and a polychromator, combined with a laser scanning confocal microscope and a multi-dimensional Time Correlated Single Photon Counting (TCSPC) electronic apparatus. Although it represents an advancement and gives experimental confirmations of the interest of the sFLIM approach, it has two basic drawbacks. First, the photon detection efficiency of the PMT detector is inherently low. Second, the TCSPC electronics allows to acquire information for only one spectral channel at a time, thus limiting the data acquisition speed (see Sect 1.2 for a more extended explanation). There is a strong potential in the market for a compact and especially 10x faster sFLIM instrumentation system that can overcome such limitations.

  • Status
  • Completed
  • Project Launch
  • 01 January 2009
  • Project completed
  • 01 December 2012
molecular machines biological processes pathos physiology

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