New photonic imaging systems for biomedical applications
University College Cork
Oxygen (O2) is one of the essential environmental parameters for living organisms, as it plays a vital role in their metabolism. Therefore, monitoring the distribution of O2 and its dynamics is essential for physiological studies. Assessment of tissue oxygenation by means of phosphorescent probes and optical microscopy has been increasingly used in biological and medical research. O2 imaging by PLIM is gaining popularity as it can provide an accurate, quantitative, and calibration-free mapping of O2 concentration in complex biological samples and is unaffected by external factors such as probe concentration, autofluorescence, photobleaching, optical alignment, excitation intensity, and scattering. Current PLIM platforms have limitations in image acquisition speed, sensitivity, penetration depth, accuracy, and general usability. TCSPC with raster scanning technique is frequently used in PLIM, but it is slow as it requires long pixel dwell times to measure long-lived phosphorescence. Other commonly used techniques include gated CCD and CMOS cameras, which lack single photon sensitivity and have a low frame rate. Moreover, the majority of the current PLIM systems use a microscopic format, while macroscopic systems are rarely available. We demonstrate a new macro-imager that overcomes many of these limitations. The imager is based on the new time-stamping Tpx3Cam optical camera, which combines a novel silicon optical sensor and a Timepix3 readout chip that has the capability to record the TOA and TOT at each pixel simultaneously. For TCSPC-PLIM applications, the Tpx3Cam imager was built by integrating the camera with an image intensifier that provided single photon sensitivity, an emission filter, and a macro lens using a Cricket adapter. Sample excitation was performed with an LED controlled by a pulse generator and synchronised with the camera. A custom-designed software was used to acquire the Tpx3Cam raw data in a binary format, and a C-language code was used to post-process the data. The resulting data matrix was then fitted with a bi-exponential function to determine the lifetime values. Planar phosphorescent O2 sensors and a resolution plate mask were used to characterize the imager. The performance and resolution of the imager were optimized with different image processing techniques, and the spatial and temporal resolution achieved were adequate for wide-field PLIM applications. Various commercial and non-commercial O2-sensitive phosphorescent materials were evaluated with the new imager. The lifetime signals were recorded for 20 s and the PLIM images generated by the imager showed good uniformity and lifetime contrast between the different materials with varying temperature and oxygenation conditions. The phosphorescence lifetime values were also consistent with those measured by alternative methods. Next, we demonstrated the application of the imager for mapping O2 concentration in live tissue samples stained with a PtBP-based NanO2-IR probe. The tissues were stained passively or by microinjections, and PLIM was performed under resting conditions and upon inhibition of respiration. The imager allowed accurate phosphorescence lifetime measurement of the probe and generated detailed O2 concentration maps of the tissue surface and even at depths of up to 0.5 mm inside the tissue. Lastly, we demonstrated the Tpx3Cam imager in model in vivo applications to map O2 concentration and tissue hypoxia in grafted tumours of laboratory animals. The probe delivery conditions in the animals were optimized to prevent adverse effects and enable long-term detection of the probe in the grafts. The live animals were injected with CT26 cells stained with the NanO2-IR probe to develop tumours with characteristic phosphorescence. The animals were monitored, and tumours were allowed to grow for 3, 7, 10 and 17 days. O2 imaging of live and euthanized animals at different time points produced phosphorescence lifetime values that reflected the hypoxic state of the tissue. Confocal PLIM microscopy was also performed on the excised tissues to demonstrate the distribution of the probe in the tumour. These results demonstrate that the new Tpx3Cam imager shows promising performance in PLIM mode with high spatial resolution. It can image objects up to several centimetres in size with flexible optical alignment (e.g., vertical or horizontal configuration). It can produce accurate lifetime values at a superior speed and sensitivity that can be converted to O2 concentration maps. The application of the imager ranges from the characterization of solid-state O2-sensors to O2 imaging of sizable biological samples, including respiring cells, live post-mortem animal tissues and organs and in vivo imaging of tissue hypoxia in animal models. Overall, the imager can be used for quantitative wide-field PLIM in physiological in vivo studies in a minimally invasive manner.
Oxygen sensing and imaging , Fluorescence and phosphorescence lifetime imaging , PLIM , FLIM , Time correlated single photon counting , TCSPC , Tpx3Cam camera
Sen, R. 2023. New photonic imaging systems for biomedical applications. PhD Thesis, University College Cork.