Klaus Kemnitz

Homo-FRET Microscopy in Living Cells to Measure Monomer-Dimer Transition of GFP-Tagged Proteins

Fluorescence anisotropy decay microscopy was used to determine, in individual living cells, the spatial monomer-dimer distribution of proteins, as exemplified by herpes simplex virus thymidine kinase (TK) fused to green fluorescent protein (GFP). Accordingly, the fluorescence anisotropy dynamics of two fusion proteins (TK27GFP and TK366GFP) was recorded in the confocal mode by ultra-sensitive time-correlated single-photon counting. This provided a measurement of the rotational time of these proteins, which, by comparing with GFP, allowed the determination of their oligomeric state in both the cytoplasm and the nucleus. It also revealed energy homo-transfer within aggregates that TK366GFP progressively formed. Using a symmetric dimer model, structural parameters were estimated; the mutual orientation of the transition dipoles of the two GFP chromophores, calculated from the residual anisotropy, was 44.6 +/- 1.6 degrees, and the upper intermolecular limit between the two fluorescent tags, calculated from the energy transfer rate, was 70 A. Acquisition of the fluorescence steady-state intensity, lifetime, and anisotropy decay in the same cells, at different times after transfection, indicated that TK366GFP was initially in a monomeric state and then formed dimers that grew into aggregates. Picosecond time-resolved fluorescence anisotropy microscopy opens a promising avenue for obtaining structural information on proteins in individual living cells, even when expression levels are very low.

Picosecond-Hetero-FRET Microscopy to Probe Protein-Protein Interactions in Live Cells

By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG.

Homo-FRET versus hetero-FRET to probe homodimers in living cells.

Publisher Summary The purpose of this chapter is to provide information on the homo-fluorescence resonance energy transfer (FRET) versus hetero-FRET to probe homodimers in living cells. FRET is a nonradiative phenomenon in which energy is transferred from a donor fluorophore to an acceptor chromophore with an efficiency that depends on the distance between the two chromophores, the extent of overlap between the donor emission and acceptor excitation spectra, the quantum yield of the donor, and the relative orientation of the donor and acceptor. For homo-FRET, because the photophysical properties of the two donor molecules are the same, the excitation energy is reversibly transferred between the fluorescent tags. Time-resolved fluorescence anisotropy monitors any process that changes the polarization of the emitted fluorescence during the excited state. Consequently, the fluorescence anisotropy decay depends on (1) rotational movements of the fluorescent molecules and (2) energy transfer taking place within the fluorescence time scale. In addition, according to the type of interaction, hetero-or homodimer, the methodology, hetero- or homo-FRET, must be judiciously chosen to obtain the best information about structural data within the macromolecular complex.

Restrained torsional dynamics of nuclear DNA in living proliferative mammalian cells.

Physical parameters, describing the state of chromatinized DNA in living mammalian cells, were revealed by in situ fluorescence dynamic properties of ethidium in its free and intercalated states. The lifetimes and anisotropy decays of this cationic chromophore were measured within the nuclear domain, by using the ultra-sensitive time-correlated single-photon counting technique, confocal microscopy, and ultra-low probe concentrations. We found that, in living cells: 1) free ethidium molecules equilibrate between extracellular milieu and nucleus, demonstrating that the cation is naturally transported into the nucleus; 2) the intercalation of ethidium into chromatinized DNA is strongly inhibited, with relaxation of the inhibition after mild (digitonin) cell treatment; 3) intercalation sites are likely to be located in chromatin DNA; and 4) the fluorescence anisotropy relaxation of intercalated molecules is very slow. The combination of fluorescence kinetic and fluorescence anisotropy dynamics indicates that the torsional dynamics of nuclear DNA is highly restrained in living cells.

Picosecond time-resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein

Time‐resolved microspectrofluorometry in live cells, based on time‐ and space‐correlated single‐photon counting, is a novel method to acquire spectrally resolved fluorescence decays, simultaneously in 256 wavelength channels. The system is calibrated with a full width at half maximum (FWHM) of 90 ps for the temporal resolution, a signal‐to‐noise ratio of 106, and a spectral resolution of 30 (Δλ/Λ). As an exemple, complex fluorescence dynamics of ethidium and cyan fluorescent protein (CFP) in live cells are presented. Free and DNA intercalated forms of ethidium are simultaneously distinguishable by their relative lifetime (1.7 ns and 21.6 ns) and intensity spectra (shift of 7 nm). By analysing the complicated spectrally resolved fluorescence decay of CFP, we propose a fluorescence kinetics model for its excitation/desexcitation process. Such detailed studies under the microscope and in live cells are very promising for fluorescence signal quantification.

Novel detectors for fluorescence lifetime imaging on the picosecond time scale

Simultaneous acquisition of time and space information on the picosecond time scale became feasible with a recent advance in microchannel-plate photomultiplier-tube (MCP-PMT) technology: we present two novel MCP-PMT detectors for time- and space-correlated single-photon counting (TSCSPC), featuring a space-sensitive delay-line (DL) anode and quadrant anode (QA), respectively. The linear DL-MCP-PMT is characterized by a spatial instrument response function (IRF) of 100-Μm FWHM, resulting in 200 space channels, whereas the QA-MCP-PMT is a 2D imager with 400 x 400 pixels at 40-Μm resolution. The detectors have a temporal IRF of 75 ps (DL) and 80 ps (QA) FWHM, sufficient for 10 ps time resolution, at a dynamic range of 105 of the uncooled detector. A throughput of 105 cps is possible; in the imaging mode without timing, the QA-detector can achieve 106 cps. We present time-resolved spectroscopy of DNA probes (DAPI, TOTO, C350) in solution, in micelles, complexed to DNA, protein, and fixed cells. Aging of DAPI stock solutions is reported. A polarity model for the photophysics of DAPI is proposed. First microscope lifetime images on the picosecond time scale show a clear potential for dynamic stray-light rejection and kinetic discrimination of probe-protein and probe-DNA complexes.

Time- and Space-Correlated Single Photon Counting Spectroscopy

Simultaneous acquisition of time- and space-information in time-domain single photon counting spectroscopy became feasible by a recent advance in microchannel-plate photomultiplier-tube technology: we present a novel MCP-PMT detector, featuring a space- sensitive delay-line anode. The detector is characterized by temporal and spatial instrument response functions of 75 ps and 100 micrometer FWHM, respectively, at 200 space channels and a dynamic range of 105. By employing a two-dimensional multichannel analyzer with transputer, 70.000 cps through-put or higher is possible. No photons are lost at the exit slit of the monochromator, as in standard, one-channel time-correlated single photon counting spectroscopy, and sensitive biological samples can be studied at reduced excitation energies. We applied the novel detector to study the basic photophysics of DAPI and its interaction with DNA.

Fluorescence lifetime imaging of cells on the picosecond timescale

Novel MCP detectors for time- and space-correlated single photon counting (TSCSPC) spectroscopy, featuring delay-line (DL) or quadrant anode (QA), are employed in microscopic fluorescence lifetime imaging on the picosecond time scale. The linear DL-MCP-PMT is characterized by a spatial instrument response function (IRF) of 100 micrometer FWHM, resulting in 200 space channels, whereas the QA-MCP-PMT is a 2D imager with 400 by 400 pixel at 40 micrometer resolution. The detectors have a temporal IRF of 75 ps (DL) and 120 ps (QA) FWHM, sufficient for 10 ps time resolution. First results on TOTO-fixed cell systems are presented, demonstrating high-quality kinetics at subcellular resolution, with up to 6 lifetime species at higher dye concentrations, characteristically distributed among individual cell compartments. A comparison with TOTO/DNA- suspensions is made that serve as reference system.

Two-stimuli manipulation of a biological motor

F1-ATPase is an enzyme acting as a rotary nano-motor. During catalysis subunits of this enzyme complex rotate relative to other parts of the enzyme. Here we demonstrate that the combination of two input stimuli causes stop of motor rotation. Application of either individual stimulus did not significantly influence motor motion. These findings may contribute to the development of logic gates using single biological motor molecules.

Ultrasensitive detection of cholecystokinin (CCK) by laser-induced time-resolved fluorescence diagnostics

An ultrasensitive detection method of CCK in aqueous solution was developed. This method utilizes intrinsic native fluorescence properties of CCK-molecules. A new delay-line multichannel plate photomultiplier (MCP PMT) was used to measure both time and wavelength resolved fluorescence properties simultaneously. Both CCK4 and CCK8 showed biexponential fluorescence decay. While the short lived fluorescence decay time components were rather similar, the long-lived components differed by about 1 ns. In the case of CCK8 the long-lived component was red-shifted by 30 nm. Based on these photophysical data an experimental setup for an ultrasensitive detection was developed. The lowest detection limit was achieved by investigating the fluorescence intensity within a time gate (gate duration 2 ns) in the fluorescence maximum. In combination with a confocal setup, using a Cassegrainean configuration, CCK could be detected up to 10-12 M.