Large-area spectrally encoded confocal endomicroscopy of the human esophagus in vivo.

Background And Objective: Diagnosis of esophageal diseases is often hampered by sampling errors that are inherent in endoscopic biopsy, the standard of care. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal endomicroscopy technology that has the potential to visualize cellular features from large regions of the esophagus, greatly decreasing the likelihood of sampling error. In this paper, we report results from a pilot clinical study imaging the human esophagus in vivo with a prototype SECM endoscopic probe.

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.

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.

Endoscopic probe optics for spectrally encoded confocal microscopy.

Spectrally encoded confocal microscopy (SECM) is a form of reflectance confocal microscopy that can achieve high imaging speeds using relatively simple probe optics. Previously, the feasibility of conducting large-area SECM imaging of the esophagus in bench top setups has been demonstrated. Challenges remain, however, in translating SECM into a clinically-useable device; the tissue imaging performance should be improved, and the probe size needs to be significantly reduced so that it can fit into luminal organs of interest.

A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization.

Germline missense mutations affecting a single BRCA2 allele predispose humans to cancer. Here we identify a protein-targeting mechanism that is disrupted by the cancer-associated mutation, BRCA2(D2723H), and that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic.

Design and application of a confocal microscope for spectrally resolved anisotropy imaging.

Biophysical imaging tools exploit several properties of fluorescence to map cellular biochemistry. However, the engineering of a cost-effective and user-friendly detection system for sensing the diverse properties of fluorescence is a difficult challenge. Here, we present a novel architecture for a spectrograph that permits integrated characterization of excitation, emission and fluorescence anisotropy spectra in a quantitative and efficient manner.

A FRET sensor for non-invasive imaging of amyloid formation in vivo.

Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins.

Quantitative fluorescence microscopy techniques.

Fluorescence microscopy is a non-invasive technique that allows high resolution imaging of cytoskeletal structures. Advances in the field of fluorescent labelling (e.g.

FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells.

Background: During its intraerythrocytic asexual reproduction cycle Plasmodium falciparum consumes up to 80% of the host cell hemoglobin, in large excess over its metabolic needs. A model of the homeostasis of falciparum-infected red blood cells suggested an explanation based on the need to reduce the colloid-osmotic pressure within the host cell to prevent its premature lysis. Critical for this hypothesis was that the hemoglobin concentration within the host cell be progressively reduced from the trophozoite stage onwards.

Group refractive index reconstruction with broadband interferometric confocal microscopy.

We propose a novel method of measuring the group refractive index of biological tissues at the micrometer scale. The technique utilizes a broadband confocal microscope embedded into a Mach-Zehnder interferometer, with which spectral interferograms are measured as the sample is translated through the focus of the beam. The method does not require phase unwrapping and is insensitive to vibrations in the sample and reference arms.

Theoretical investigation of the photon efficiency in frequency-domain fluorescence lifetime imaging microscopy.

We investigate the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy, using both theoretical and Monte Carlo methods. Our analysis differs from previous work in that it incorporates the data fitting process used in real experiments, allows for the arbitrary choice of excitation and gain waveforms, and calculates lifetimes as well as associated F-values from higher harmonics in the data. Using our analysis, we found different photon efficiencies to those previously reported and were able to propose optimal excitation and gain waveforms.

Nonparaxial vector-field modeling of optical coherence tomography and interferometric synthetic aperture microscopy.

A large-aperture, electromagnetic model for coherent microscopy is presented and the inverse scattering problem is solved. Approximations to the model are developed for near-focus and far-from-focus operations. These approximations result in an image-reconstruction algorithm consistent with interferometric synthetic aperture microscopy (ISAM): this validates ISAM processing of optical-coherence-tomography and optical-coherence-microscopy data in a vectorial setting.

High-resolution three-dimensional imaging of biofilm development using optical coherence tomography.

We describe the use of optical coherence tomography (OCT) for high-resolution, real-time imaging of three-dimensional structure and development of a Pseudomonas aeruginosa biofilm in a standard capillary flow-cell model. As the penetration depth of OCT can reach several millimeters in scattering samples, we are able to observe complete biofilm development on all surfaces of a 1 mm x 1 mm flow-cell. We find that biofilm growing at the bottom of the tube has more structural features including voids, outward projections, and microcolonies while the biofilm growing on the top of the tube is relatively flat and contains less structural features.

In vivo detection of exercised-induced ultrastructural changes in genetically-altered murine skeletal muscle using polarization-sensitive optical coherence tomography.

Skeletal muscle fibers are a known source of form birefringence in biological tissue. The birefringence present in skeletal muscle is associated with the ultrastructure of individual sarcomeres, specifically the arrangement of A-bands corresponding to the thick myosin filaments. Certain structural proteins that prevent damage and maintain the structural and functional health of the muscle fiber preserve the organization of the Abands in skeletal muscle.