Alicia Megia-Fernandez

Time-resolved spectroscopy at 19,000 lines per second using a CMOS SPAD line array enables advanced biophotonics applications

A SPAD-based line sensor fabricated in 130 nm CMOS technology capable of acquiring time-resolved fluorescence spectra (TRFS) in 8.3 milliseconds is presented. To the best of our knowledge, this is the fastest time correlated single photon counting (TCSPC) TRFS acquisition reported to date. The line sensor is an upgrade to our prior work and incorporates: i) parallelized interface from sensor to surrounding circuitry enabling high line rate to the PC (19,000 lines/s) and ii) novel time-gating architecture where detected photons in the OFF region are rejected digitally after the output stage of the SPAD. The time-gating architecture was chosen to avoid electrical transients on the SPAD high voltage supplies when gating is achieved by excess bias modulation. The time-gate has an adjustable location and time window width allowing the user to focus on time-events of interest. On-chip integrated center-of-mass (CMM) calculations provide efficient acquisition of photon arrivals and direct lifetime estimation of fluorescence decays. Furthermore, any of the SPC, TCSPC and on-chip CMM modes can be used in conjunction with the time-gating. The higher readout rate and versatile architecture greatly empower the user and will allow widespread applications across many techniques and disciplines. Here we focused on 3 examples of TRFS and time-gated Raman spectroscopy: i) kinetics of chlorophyll A fluorescence from an intact leaf; ii) kinetics of a thrombin biosensor FRET probe from quenched to fluorescence states; iii) ex vivo mouse lung tissue autofluorescence TRFS; iv) time-gated Raman spectroscopy of toluene at 3056 cm-1 peak. To the best of our knowledge, we detect spectrally for the first time the fast rise in fluorescence lifetime of chlorophyll A in a measurement over single fluorescent transient.

Peptides for optical medical imaging and steps towards therapy

Optical medical imaging is a rapidly growing area of research and development that offers a multitude of healthcare solutions both diagnostically and therapeutically. In this review, some of the most recently described peptide-based optical probes are reviewed with a special emphasis on their in vivo use and potential application in a clinical setting.

In situ identification of Gram-negative bacteria in human lungs using a topical fluorescent peptide targeting lipid A

A topically administered fluorescently labeled peptide targeting lipid A permits rapid, real-time visualization of bacteria in the distal human lung. Lung infection in real time Lung infections are frequent causes of complications in mechanically ventilated and immunosuppressed patients. However, the diagnosis is challenging, requires risky procedures, and is time consuming. Now, Akram et al. have developed an imaging method that is able to detect Gram-negative bacteria in real time in the distal part of the human lung. Using a fluorescent probe binding to lipid A, a molecule expressed on Gram-negative bacterial membranes, in combination with an optical endomicroscope, the researchers rapidly detected Gram-negative infections in distal airways in hospitalized individuals. The results suggest that the approach could accelerate the diagnosis of bacterial lung infection and facilitate the evaluation of antibiotic treatment efficacy. Respiratory infections in mechanically ventilated patients caused by Gram-negative bacteria are a major cause of morbidity. Rapid and unequivocal determination of the presence, localization, and abundance of bacteria is critical for positive resolution of the infections and could be used for patient stratification and for monitoring treatment efficacy. Here, we developed an in situ approach to visualize Gram-negative bacterial species and cellular infiltrates in distal human lungs in real time. We used optical endomicroscopy to visualize a water-soluble optical imaging probe based on the antimicrobial peptide polymyxin conjugated to an environmentally sensitive fluorophore. The probe was chemically stable and nontoxic and, after in-human intrapulmonary microdosing, enabled the specific detection of Gram-negative bacteria in distal human airways and alveoli within minutes. The results suggest that pulmonary molecular imaging using a topically administered fluorescent probe targeting bacterial lipid A is safe and practical, enabling rapid in situ identification of Gram-negative bacteria in humans.

Rapid Polymer Conjugation Strategies for the Generation of pH-Responsive, Cancer Targeting, Polymeric Nanoparticles

The combination of controlled living polymerization in association with rapid and highly efficient macromolecule conjugation strategies provides a powerful tool for the synthesis of novel polymeric materials. Here functional block copolymers were rapidly and quantitatively conjugated using an efficient reaction between polymers containing a phenolic group and the 4-phenyl-3 H-1,2,4-triazole-3,5(4 H)-dione (PTAD) moiety and used to generate nanoparticles that encapsulated drugs. pH responsive amphiphilic block copolymers, which self-assemble into nanoparticles, were fabricated using our novel polymer conjugation strategy with the resulting system designed to promote drug release within the acidic milieu of the cancer microenvironment. The conjugation strategy also enabled the direct tagging of the nanoparticles with a range of fluorophores, targeting assets, or both with cargo release demonstrated in cancer cells.

Multiplexed fibre optic sensing in the distal lung (Conference Presentation)

We present a toolkit for a multiplexed pH and oxygen sensing probe in the distal lung using multicore fibres. Measuring physiological relevant parameters like pH and oxygen is of significant importance in understanding changes associated with disease pathology. We present here, a single multicore fibre based pH and oxygen sensing probe which can be used with a standard bronchoscope to perform in vivo measurements in the distal lung. The multiplexed probe consists of fluorescent pH sensors (fluorescein based) and oxygen sensors (Palladium porphyrin complex based) covalently bonded to silica microspheres (10 µm) loaded on the distal facet of a 19 core (10 µm core diameter) multicore fibre (total diameter of ~150 µm excluding coating). Pits are formed by selectively etching the cores using hydrofluoric acid, multiplexing is achieved through the self-location of individual probes on differing cores. This architecture can be expanded to include probes for further parameters. Robust measurements are demonstrated of self-referencing fluorophores, not limited by photobleaching, with short (100ms) measurement times at low (~10µW) illumination powers. We have performed on bench calibration and tests of in vitro tissue models and in an ovine whole lung model to validate our sensors. The pH sensor is demonstrated in the physiologically relevant range of pH 5 to pH 8.5 and with an accuracy of ± 0.05 pH units. The oxygen sensor is demonstrated in gas mixtures downwards from 20% oxygen and in liquid saturated with 20% oxygen mixtures (~8mg/L) down to full depletion (0mg/L) with ~0.5mg/L accuracy.