A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.This work was funded by grants from the Medical Research Council, the Wellcome Trust, the Alzheimer Research UK Trust, and the Engineering and Physical Sciences Research Council. W.Y.C. is funded by a China Scholarship Council-Cambridge Scholarship. D.R. is a Principal Research Fellow of the Wellcome Trust.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0006349515000752#

State-resolved valence shell photoionization of Be-like ions: experiment and theory

High-resolution photoionization experiments were carried out using beams of Be-like C$^{2+}$, N$^{3+}$, and O$^{4+}$ ions with roughly equal populations of the $^1$S ground-state and the $^3$P$^o$ manifold of metastable components. The energy scales of the experiments are calibrated with uncertainties of 1 to 10 meV depending on photon energy. Resolving powers beyond 20,000 were reached allowing for the separation of contributions from the individual metastable $^3$P$^o_0$, $^3$P$^o_1$, and $^3$P$^o_2$ states. The measured data compare favourably with semi-relativistic Breit-Pauli R-matrixComment: 23 figures and 3 table

Comprehensive confocal endomicroscopy of the esophagus in vivo

Background and study aims: Biopsy sampling error can be a problem for the diagnosis of certain gastrointestinal tract diseases. Spectrally-encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has the potential to overcome sampling error by imaging large regions of gastrointestinal tract tissues. The aim of this study was to test a recently developed SECM endoscopic probe for comprehensively imaging large segments of the esophagus at the microscopic level in vivo. Methods: Topical acetic acid was endoscopically applied to the esophagus of a normal living swine. The 7 mm diameter SECM endoscopic probe was transorally introduced into the esophagus over a wire. Optics within the SECM probe were helically scanned over a 5 cm length of the esophagus. Confocal microscopy data was displayed and stored in real time. Results: Very large confocal microscopy images (length = 5 cm; circumference = 2.2 cm) of swine esophagus from three imaging depths, spanning a total area of 33 cm2, were obtained in about 2 minutes. SECM images enabled the visualization of cellular morphology of the swine esophagus, including stratified squamous cell nuclei, basal cells, and collagen within the lamina propria. Conclusions: The results from this study suggest that the SECM technology can rapidly provide large, contiguous confocal microscopy images of the esophagus in vivo. When applied to human subjects, the unique comprehensive, microscopic imaging capabilities of this technology may be utilized for improving the screening and surveillance of various esophageal diseases

The potential of optical proteomic technologies to individualize prognosis and guide rational treatment for cancer patients

Genomics and proteomics will improve outcome prediction in cancer and have great potential to help in the discovery of unknown mechanisms of metastasis, ripe for therapeutic exploitation. Current methods of prognosis estimation rely on clinical data, anatomical staging and histopathological features. It is hoped that translational genomic and proteomic research will discriminate more accurately than is possible at present between patients with a good prognosis and those who carry a high risk of recurrence. Rational treatments, targeted to the specific molecular pathways of an individual’s high-risk tumor, are at the core of tailored therapy. The aim of targeted oncology is to select the right patient for the right drug at precisely the right point in their cancer journey. Optical proteomics uses advanced optical imaging technologies to quantify the activity states of and associations between signaling proteins by measuring energy transfer between fluorophores attached to specific proteins. Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) assays are suitable for use in cell line models of cancer, fresh human tissues and formalin-fixed paraffin-embedded tissue (FFPE). In animal models, dynamic deep tissue FLIM/FRET imaging of cancer cells in vivo is now also feasible. Analysis of protein expression and post-translational modifications such as phosphorylation and ubiquitination can be performed in cell lines and are remarkably efficiently in cancer tissue samples using tissue microarrays (TMAs). FRET assays can be performed to quantify protein-protein interactions within FFPE tissue, far beyond the spatial resolution conventionally associated with light or confocal laser microscopy. Multivariate optical parameters can be correlated with disease relapse for individual patients. FRET-FLIM assays allow rapid screening of target modifiers using high content drug screens. Specific protein-protein interactions conferring a poor prognosis identified by high content tissue screening will be perturbed with targeted therapeutics. Future targeted drugs will be identified using high content/throughput drug screens that are based on multivariate proteomic assays. Response to therapy at a molecular level can be monitored using these assays while the patient receives treatment: utilizing re-biopsy tumor tissue samples in the neoadjuvant setting or by examining surrogate tissues. These technologies will prove to be both prognostic of risk for individuals when applied to tumor tissue at first diagnosis and predictive of response to specifically selected targeted anticancer drugs. Advanced optical assays have great potential to be translated into real-life benefit for cancer patients