Acceleration of the ICTM image restoration algorithm

We have implemented a simple modification of the ICTM image restoration algorithm that reduces the computational requirements considerably. The modified algorithm converges to the same solution as alternative approaches for computing non-negatively constrained Tikhonov solutions. It compares favourably with these algorithms in terms of speed.     

The role of photon statistics in fluorescence anisotropy imaging.

Anisotropy imaging can be used to image resonance energy transfer between pairs of identical fluorophores and, thus, constitutes a powerful tool for monitoring protein homo-association in living single cells. The requirement for only a single fluorophore significantly simplifies biological preparation and interpretation. We use quantitative methods for the acquisition and image processing of anisotropy data that return the expected error of the anisotropy per pixel based on photon statistics. The analysis methods include calibration procedures and allow for a balance in spatial, anisotropy, and temporal resolution. They are featured here with anisotropy images of fluorescent calibration beads and enhanced green fluorescent protein complexes in live cells.     

Actinomycin D binding to single-stranded DNA: sequence specificity and hemi-intercalation model from fluorescence and 1H NMR spectroscopy

Various in vitro studies have shown that delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, has a variety of inhibitory effects on immune functions including effects on macrophages. The present studies have examined the mechanism of THC's effects on tumor necrosis factor alpha (TNF-alpha), a major macrophage-produced cytokine and an important mediator involved in cytokine networks and in host defense mechanisms. Exposure of macrophages to medium containing THC has resulted in low levels of soluble TNF-alpha protein and reduced TNF-alpha bioactivity in the culture supernatant. However, THC did not inhibit the levels of LPS-induced TNF-alpha mRNA and intracellular TNF-alpha precursor protein, had only a weak effect on expression of membrane-bound TNF-alpha, but suppressed TNF-alpha maturation/secretion by macrophages. The higher the THC concentration in the medium during TNF-alpha induction, the greater the amount of intracellular TNF-alpha precursors that accumulated in the activated macrophages and the less mature TNF-alpha was released from the cells. Data suggest that TNF-alpha production by macrophages was altered greatly by exposure to THC at the levels of TNF-alpha precursor maturation and secretion.     

Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation

The aggregation states of the epidermal growth factor receptor (EGFR) on single A431 human epidermoid carcinoma cells were assessed with two new techniques for determining fluorescence resonance energy transfer: donor photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence lifetime imaging microscopy (FLIM). Fluorescein-(donor) and rhodamine-(acceptor) labeled EGF were bound to the cells and the extent of oligomerization was monitored by the spatially resolved FRET efficiency as a function of the donor/acceptor ratio and treatment conditions. An average FRET efficiency of 5% was determined after a low temperature (4 degrees C) incubation with the fluorescent EGF analogs for 40 min. A subsequent elevation of the temperature for 5 min caused a substantial increase of the average FRET efficiency to 14% at 20 degrees C and 31% at 37 degrees C. In the context of a two-state (monomer/dimer) model for the EGFR, these FRET efficiencies were consistent with minimal average receptor dimerizations of 13, 36, and 69% at 4, 20, and 37 degrees C, respectively. A431 cells were pretreated with the monoclonal antibody mAb 2E9 that specifically blocks EGF binding to the predominant population of low affinity EGFR (15). The average FRET efficiency increased dramatically to 28% at 4 degrees C, indicative of a minimal receptor dimerization of 65% for the subpopulation of high affinity receptors. These results are in accordance with prior studies indicating that binding of EGF leads to a fast and temperature-dependent microclustering of EGFR, but suggest in addition that the high affinity functional subclass of receptors on quiescent A431 cells are present in a predimerized or oligomerized state. We propose that the transmission of the external ligand-binding signal to the cytoplasmic domain is effected by a concerted relative rotational rearrangement of the monomeric units comprising the dimeric receptor, thereby potentiating a mutual activation of the tyrosine kinase domains.     

Interferometric measurement of fluorescence excitation spectra

A Michelson interferometer has been adapted as an excitation source for fluorescence spectroscopy. A moving fringe pattern was generated by linear displacement of the movable mirror of the Michelson interferometer coupled to a xenon-arc lamp. This spectrally modulated source was monitored by a reference photomultiplier and used for exciting a Rhodamine B solution. The fluorescence emission at >645 nm was detected by a second photomultiplier. The two interferograms were acquired by a dual-channel digital oscilloscope, and their discrete Fourier transforms and corresponding power spectra were generated in a computer. The power spectrum of the emission signal represented the excitation spectrum, as was confirmed by comparison with the absorption spectrum of Rhodamine B. Thisoptical arrangement is well suited for acquiring fluorescence excitation spectra in the optical microscopy of biological specimens.     

Fluorescence correlation microscopy (FCM)-fluorescence correlation spectroscopy (FCS) taken into the cell

Confocal fluorescence correlation spectroscopy (FCS) and other confocal spectroscopic techniques are ideally suited for the analysis of molecular interactions at the subcellular level. However, one requires exact positioning in three dimensions within the cell. Our instrument integrates FCS with high sensitivity digital imaging microscopy and high precision positioning. We present first measurements of intracellular FCS, with specification of the instrumental requirements and methods of data analysis. We propose the term fluorescence correlation microscopy (FCM) for this extended modality of FCS.     

Determination of DNA helical handedness by fluorescence resonance energy transfer

Fluorescence resonance energy transfer (FRET) has been used to determine the helical handedness, twist and rise of different DNA conformations. The approach is based on the construction of a set of molecules consisting of two fused helical segments, one of which is in a known reference helical form. The duplexes are covalently labeled at one end with a donor and at the other with an acceptor. By systematically shifting the position of the junction while maintaining constant the total length in base-pairs, the variation in the efficiency of energy transfer can be shown to depend primarily and sensitively on the differences in helical twist and rise of the two constituent segments. If the latter have the same helical sense, one predicts a FRET signal that is a monotonic function of the junctional position. In contrast, a periodic function arises when two segments are of opposite handedness. The formalism includes explicit consideration of dye orientation (the dipole-dipole orientation factor kappa) and an implementation valid for single helix molecules, and introduces new functions of measured fluorescence signals for establishing the FRET efficiency. The method has been applied to a family of oligonucleotides forming hairpin duplexes containing an antiparallel-stranded (aps) d(m5C.G)m segment labeled at the 5' end with fluorescein (donor) and a second parallel- stranded d(A.T)N-m segment (psAt-DNA) labeled at the hairpin loop with the sulfoindocyanine dye Cy3. The segment lengths were in the range 4 to 12, but the total length N was maintained constant at 16. The d(m5C.G) sequence was chosen due to its capacity for adopting a B or a Z conformation at low and high concentrations of salt, respectively. The parallel-stranded d(A.T) sequence served as the second segment in order to determine the helical rise and twist of psAT-DNA, presumed to be right-handed from molecular modeling and a prior study of topologically constrained DNA. A Z-DNA/ps-DNA junction was created between the two segments by inducing a B-Z transition in d(m5C.G)m with MgCl2. The range of required salt concentration was established by circular dichroism measurements. FRET efficiency values of 0.38 to 0.41 were obtained for the oligonucleotides with the d(m5C.G) segment in the B conformation. In contrast, upon induction of the B-Z transition the FRET efficiency was a decreasing function of the d(m5C.G) content (0.38 to 0.28 for m = 6 to 12). Helical parameters were estimated from functional fits of the data, and were consistent with the known properties of B- and Z-DNAs and with the conclusion that psAT-DNA has a helical rise and twist close to that of B-DNA. The approach outlined here is not restricted to DNA but can be applied to other helical structures, e.g. RNA, proteins, and protein-nucleic acid complexes.     

Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin

The subcellular localization and corresponding quaternary state of fluorescent labelled cholera toxin were determined at different time points after exposure to living cells by a novel form of fluorescence confocal microscopy. The compartmentalization and locus of separation of the pentameric B subunits (CTB) from the A subunit (CTA) of the toxin were evaluated on a pixel-by-pixel (voxel-by-voxel) basis by measuring the fluorescence resonance energy transfer (FRET) between CTB labelled with the sulfoindocyanine dye Cy3 and an antibody against CTA labelled with Cy5. The FRET efficiency was determined by a new technique based on the release of quenching of the Cy3 donor after photodestruction of the Cy5 acceptor in a region of interest within the cell. The results demonstrate vesicular transport of the holotoxin from the plasma membrane to the Golgi compartment with subsequent separation of the CTA and CTB subunits. The CTA subunit is redirected to the plasma membrane by retrograde transport via the endoplasmic reticulum whereas the CTB subunit persists in the Golgi compartment.