Tushar R. Choudhary

Assessment of acute mild hypoxia on retinal oxygen saturation using snapshot retinal oximetry

PURPOSE To study the effect of acute mild hypoxia on retinal oxygen saturation. METHODS Spectral retinal images were acquired under normoxic and hypoxic conditions for 10 healthy human volunteers (six male, four female, aged 25 ± 5 years [mean ± SD]) using a modified fundus camera fitted with an image-replicating imaging spectrometer (IRIS). Acute, mild hypoxia was induced by changing the oxygen saturation of inhaled air from 21% to 15% using a hypoxia generator with subjects breathing the hypoxic gas mixture for 10 minutes. Peripheral arterial oxygen saturation of the subjects was monitored using fingertip-pulse oximetry. Images were processed to calculate oxygen saturation, arteriovenous difference in oxygen saturation, and vessel diameter. Data are presented as mean ± SD and were analyzed using paired sample t-test with significance accepted at P < 0.05. RESULTS The retinal arterial and venous oxygen saturation was 98.5% ± 1.6% and 70.7% ± 2.7% during normoxia. A reduction in the fraction of inspired oxygen resulted in a decline (P < 0.001) in both retinal-arterial and venous oxygen saturation to 90.3% ± 2.0% and 62.4% ± 2.2%, respectively. The arteriovenous oxygen saturation difference in normoxia (27.8% ± 2.9%) and hypoxia (27.9% ± 2.1%) did not change. Retinal arteriolar and venular diameters increased (P < 0.001) by 4% and 3%, respectively, under hypoxia. CONCLUSIONS The acute inhalation of a hypoxic gas mixture resulted in a decline in both retinal-arterial and venous saturation, while arteriovenous oxygen difference was maintained with an accompanying significant increase in retinal vessel diameter. This may suggest an autoregulatory response to acute mild hypoxia.

Two-color widefield fluorescence microendoscopy enables multiplexed molecular imaging in the alveolar space of human lung tissue

Abstract. We demonstrate a fast two-color widefield fluorescence microendoscopy system capable of simultaneously detecting several disease targets in intact human ex vivo lung tissue. We characterize the system for light throughput from the excitation light emitting diodes, fluorescence collection efficiency, and chromatic focal shifts. We demonstrate the effectiveness of the instrument by imaging bacteria (Pseudomonas aeruginosa) in ex vivo human lung tissue. We describe a mechanism of bacterial detection through the fiber bundle that uses blinking effects of bacteria as they move in front of the fiber core providing detection of objects smaller than the fiber core and cladding (∼3  μm). This effectively increases the measured spatial resolution of 4  μm. We show simultaneous imaging of neutrophils, monocytes, and fungus (Aspergillus fumigatus) in ex vivo human lung tissue. The instrument has 10 nM and 50 nM sensitivity for fluorescein and Cy5 solutions, respectively. Lung tissue autofluorescence remains visible at up to 200 fps camera acquisition rate. The optical system lends itself to clinical translation due to high-fluorescence sensitivity, simplicity, and the ability to multiplex several pathological molecular imaging targets simultaneously.

Endoscopic sensing of alveolar pH

Previously unobtainable measurements of alveolar pH were obtained using an endoscope-deployable optrode. The pH sensing was achieved using functionalized gold nanoshell sensors and surface enhanced Raman spectroscopy (SERS). The optrode consisted of an asymmetric dual-core optical fiber designed for spatially separating the optical pump delivery and signal collection, in order to circumvent the unwanted Raman signal generated within the fiber. Using this approach, we demonstrate a ~100-fold increase in SERS signal-to-fiber background ratio, and demonstrate multiple site pH sensing with a measurement accuracy of ± 0.07 pH units in the respiratory acini of an ex vivo ovine lung model. We also demonstrate that alveolar pH changes in response to ventilation.

Ballistic and snake photon imaging for locating optical endomicroscopy fibres

We demonstrate determination of the location of the distal-end of a fibre-optic device deep in tissue through the imaging of ballistic and snake photons using a time resolved single-photon detector array. The fibre was imaged with centimetre resolution, within clinically relevant settings and models. This technique can overcome the limitations imposed by tissue scattering in optically determining the in vivo location of fibre-optic medical instruments.

A multifunctional endoscope for imaging, fluid delivery and fluid extraction (Conference Presentation)

We present a multifunctional endoscope capable of imaging, fluid delivery and fluid sampling in the alveolar space. The endoscope consists of an imaging fibre bundle fabricated from cost effective OM1 PCVD graded index preforms made for the telecommunications market. These low-cost fibres could potentially make our endoscope disposable after a single use. The performance of our low-cost imaging fibre bundle is shown to be comparable to the current commercial state-of-the-art. The imaging fibre bundle is packaged alongside two channels for the delivery and extraction of fluids. The fluid delivery channels can be used to deliver fluorescent smart probes for the detection of pathogens and to perform a targeted alveolar lavage without the removal of the imaging fibre as is currently standard procedure. Our endoscope is fully biocompatible and with an overall outer diameter of 1.4 mm allowing it to fit into the standard working channel of a bronchoscope. We demonstrate the use of our endoscope in ex-vivo human lungs. We show alveolar tissue and bacterial imaging over two wavelength bands 520 nm – 600 nm and 650 nm – 750 nm both commonly used for bacterial smart probe detection.

Early arriving photon imaging for locating optical endomicroscopy fibres and medical devices (Conference Presentation)

Optical fibre based endoscopes are increasingly used for imaging and sensing within the human body without navigational guidance of the miniaturised fibre probe. Meanwhile, other medical device placement is a standard procedure in clinic. We demonstrate successful imaging of optical device location with centimetre resolution in clinically relevant models, in a realistically lit environment, achieved through the detection of early arriving photons with a time resolved single photon detector array. This prototype has been developed within the UK-EPSRC Proteus project, moving advanced research technologies towards clinical implementation. Short (~100ps) laser pulses are transmitted from the tip of the endoscope at 785nm in the “optical window” where attenuation is less severe in clinical scenarios. Most of the photons that pass through tissue undergo much scattering from the disordered tissue structures providing only low accuracy determination of the location of the light source. However, some photons probabilistically undergo less scattering, travelling through the medium in an almost straight line without a much extended path. Such photons exit the body sooner than the highly scattered light. A camera based upon a 32 × 32 array of Single Photon Avalanche Diodes (SPADs) made with CMOS technology is used to image the small number photons exiting the tissue. The time resolution capabilities of such a single photon detector (50ps time bin resolution, 200ps jitter) allow observation of the photon arrival times simultaneously for all 1024 pixels of the imaging array. Photon arrival statistics distinguish the early arriving photons from the highly scattered light, revealing the endoscope location. Scattered photon arrivals peak at delays of multiple nanoseconds due to the thick tissue samples. The progression of light through complex scattering structures can be observed. Normal fluorescent room lighting has distinct emission peaks. Appropriate choice of operating wavelength between these spectral features, combined with aggressive filtering, allows operation in normal fluorescent lighting. This compact packaged system is demonstrated in a normally lit room to determine optical endomicroscope location in a whole ventilated ovine lung as well as tissue models including bone structure. At the limit of capabilities of this prototype, demonstration through an entire human torso is shown to be possible. System improvements and the potential of the next generation prototype in development will be discussed. This offers the potential for real time (sub second) imaging of device location with a portable system for application in standard medical procedures, such as catheter insertion. The avoidance of the need to confirm device placement with X-ray imaging has potential to decrease disruption to procedures throughout clinical practice.

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.

Endoscopic sensing of pH in the distal lung (Conference Presentation)

In healthy humans, the physiological state in the distal lung alveolar acinar units is tightly regulated by normal homeostatic mechanisms. Pulmonary abnormalities such as chronic obstructive pulmonary disease, that are characterized by recurrent cycles of inflammation and infection involving dense infiltration by myeloid derived peripheral blood cells, may result in significant perturbation of the homeostatic baselines of physiology in addition to host tissue damage. Therefore, the ability to quantify and monitor physiology (e.g. pH, glucose level, oxygen tension) within the alveolar acinar units would provide a key biomarker of distal lung innate defence. Although in vitro modeling of fundamental biological processes show remarkable sensitivity to physiological aberrations, little is known about the physiological state of the distal lung due to the inability to concurrently access the alveolar sacs and perform real-time sensing. Here we report on previously unobtainable measurements of alveolar pH using a fiber-optic optrode and surface enhanced Raman spectroscopy (SERS) and show that alveolar pH changes in response to ventilation. The endoscope-deployable optrode consisted of para-mercaptobenzoic acid functionalized 150 nm gold nanoshells located at the distal end, and an asymmetric dual-core optical fiber designed for spatially separated optical pump delivery and SERS signal collection in order to circumvent the unwanted Raman signal originating from the fiber itself. We demonstrate a ~ 100-fold increase in SERS signal-to-fiber background ratio and pH sensing at multiple sites in the respiratory acinar units of a whole ex vivo ovine lung model with a measurement accuracy of ± 0.07 pH units.

Towards in vivo bacterial detection in human lung(Conference Presentation)

Antibiotic resistance is a serious global concern. One way to tackle this problem is to develop new and sensitive approaches to diagnose bacterial infections and prevent unnecessary antibiotic use. With recent developments in optical molecular imaging, we are one step closer to in situ rapid detection of bacterial infections. We present here bespoke fluorescent probes for bacterial detection in ex vivo human lung tissue using fluorescence lifetime imaging microscopy (FLIM). Two in-house synthesised bespoke probes were used in this study to detect and differentiate between Gram positive and Gram negative bacterial strain using their fluorescence lifetime in the ex vivo human lung tissue. The average fluorescence lifetime of Gram positive probe (n=12) was 2.40 ± 0.25 ns and Gram negative (n=12) was 6.73 ± 0.49 ns. The human lung tissue (n=12) average fluorescence lifetime value was found to be 3.43 ± 0.19 ns. Furthermore we were also able to distinguish between dead or alive bacteria in ex vivo lung tissue based on difference in their lifetime. We have developped Fibre-FLIM methods to enable clinical translation within the Proteus Project (www.proteus.ac.uk).