The present proposal is based on the Preparatory Award for a Demonstration Project "Two-Dimensional Subnanosecond Fluorescence Lifetime Imaging of Subcellular Structures" of Area 6.1 in Biotechnology (BIO4-CT95-9253) and our Proposal No. PL960681 of 2nd Call "2D and 3D Fluorescence Lifetime Imaging of Subcellular Structures". We introduce single photon counting (SPC) picosecond FLIM (fluorescence lifetime imaging microscopy) (PFLIM) as a novel technique for 3D macro-molecular structure determination, complementary to standard techniques such as X-ray crystallography. SPC-PFLIM is an application of time and space-correlated SPC (TSCSPC) spectroscopy, distinguished by 10 ps time resolution, a dynamic range of 105, and a possible through-put of 106 cps at 400x400 pixel of 40 ,um space resolution. PFLIM has a 3D resolution limit of 3.5 A, the minimal distance of two organic probe molecules (e.g. donor-acceptor pair) in a face-to-face orientation, and is conspicuous by its in vivo potential for 3D tertiary and quarternary structure determination. In vivo 3D structure determination is possible by applying ultra-low excitation light levels and ultra-low probe concentrations (down to a single molecule or individual donor-acceptor pair). 3D structures, as traditionally obtained by, e.g., crystallography, do not always represent the macro-molecular structure of the living state. By conducting dynamic studies of electron transfer, Forster resonance energy transfer (FRET), dimerization, DNA intercalation, calcium quenching, native emission, and external quenching, we can resolve 3D structures with a resolution down to 3.5 A inside living cells.The main aim of the project is the construction of prototype 3D detection systems, based on two novel time-resolving imaging detectors: (a) the delay-line (DL)- or quadrant-anode (QA)-micro-channel plate (MCP)-PMT for TSCSPC and, as a complementary system of 100 ps resolution but shorter acquisition time, (b) the gated electron-bombarded (EB)-CCD. TSCSPC is a space-sensitive variant of standard time-correlated single photon counting (TCSPC) spectroscopy, distinguished by its unsurpassed dynamic range. Results on TSCSPC, obtained during the Preparatory Award, fully confirmed the excellent potential of these novel proprietary devices, unique on the world market. The novel EB-CCD camera system, equipped with subnanosecond gated image intensifier, will have a time resolution of about 100 ps, space resolution of 1024*1024, at < 10-7 lx (30 sec) sensitivity, and will be superior to all competing systems in the market. PFLIM is the method of choice for 3D structure determination in living cells. This will be demonstrated by work in several very different fields of cell biology: (i) structural transconformations of viral and cellular DNA inside cells or in interaction with membranes, (ii) immobilization of proteins by subcellular structures, (iii) structure-function relationship of new fusion proteins, Herpes Thymidine Kinase/Green Fluorescent Protein, in different environments, (iv) lattice-siuperlattice structural transition in lipid bilayers, and (v) mapping of cellular ion concentrations. By genetic engineering and site directed mutation, the biology groups will develop new biochemical entities (e.g., GFP-fusion proteins) of desired functions and will use the improved SPC-PFLIM technology to determine thereby its 3D tertiary and quarternary structure by applying FRET or other kinetic methods. These demonstration topics are of general significance and will be tackled by outstanding European institutes of cell biology, some of which are participating in EC Laboratory Networks and Concerted Actions, thus promising success and rapid dissemination of the new fluorescence microscope technology.
Funding SchemeCSC - Cost-sharing contracts
SW7 2AZ London