Developing New Laser Sources for Deep Tissue Penetration
The essential aspect of Amplitude is access to the 1650-1800nm wavelength. Currently, the only commercial lasers able to operate within this window are very expensive supercontinuum or OPO lasers. Amplitude is therefore developing new ultrashort fibre lasers as robust, cheap, and effective alternatives. Fibre lasers keeps the system compact and affordable, whilst the ultrashort regime reduces phototoxicity and improves image quality beyond that of current solutions.
To determine the optimal parameters for these new ultrashort pulse fibre lasers, as required by clinical imaging systems, two different sources tuneable within broad wavelength range are under development in Amplitude. The first laser covers a wavelength range between 1650-1720nm, based on nonlinear Raman effect in optical fibre. The second laser covers wavelengths between 1720-1800nm, based on hybrid mode-locking. Neither of the lasers has been developed at the wavelengths necessary to access the 1650-1800nm region, and whichever best meets Amplitude’s needs will be implemented into the final system.
To further improve the source efficiency, the chosen source will then be frequency doubled in PPLN chips, another feat not previously achieved for such lasers. This will add an additional mode (between 825nm and 900 nm for autofluorescence imaging), without the inclusion of an additional source, ensuring efficiency at the deepest design level.
Constructing Imaging Devices Suitable for the Clinic
The primary goal of Amplitude is to construct and validate a microscope, capable of extension into an endoscope, that can perform multi modal imaging in a clinical setting. Packaging the components together into these microscope/endoscope devices requires substantial effort.
To optimise any generated nonlinear signal, care must be taken during packaging about the pulse dispersion caused by the bulky optics, such as the high Numerical Aperture (NA) objectives in the microscope. Furthermore, the necessary dichroic mirrors and colour filters do not currently exist and will need to be custom made for this specific wavelength range. Task specific analysis software functions will also need to be integrated into the main software platform to enable simple comparison with histological images and a provide better visualization.
Extension of the microscope into the endoscope will also require a unique probe design. Broadband and single mode channels will carry different wavelengths, and as an endoscopic probe has much greater dispersion than a simple microscope objective, dispersion compensation units will be tailored for endoscopic deliver.
Testing Imaging Devices in the Clinic
The Amplitude microscope will also be validated and evaluated during the project, on clinical samples from patients undergoing surgery to treat bladder cancer. Samples from 100 consecutive consenting patients will be analysed label-free by the Amplitude microscope, before being sent on for definitive histopathological analysis, the current gold standard for diagnosis. By comparing Amplitude’s results to the current gold standard for bladder cancer, patterns can be identified, and thus the best options for patients determined, eventually in a much faster way.
The Amplitude endoscope testing will be more challenging. Unlike the microscope, which can be validated using ex vivo samples, the endoscope must be tested in vivo. Thus, a smaller scale of 10 consecutive consenting patients undergoing cystoscopy will provide early proof of concept. Results from this small cohort can be compared to traditional microscopy images, as well as the images from the Amplitude microscope. As a medical device, complete endoscope validation must take time well beyond the Amplitude project duration, but essential initial demonstrations will be completed during the Amplitude project.
Identifying New Biomarkers for Bladder Cancer
Amplitude also aims to improve the identification and diagnosis of tumour tissues through fast, label-free optical methods. Developing reliable biological models is the key to identify and characterise tumour biomarkers for such methods. To achieve this, pre-clinical in vitro 3D models from bladder cancer cell lines with various grade of aggressiveness/invasiveness are being generated by Amplitude to enable new insights on bladder cancer biology and behaviour.
With the assistance of the new Amplitude laser sources, these 3D models will be examined through two label-free optical techniques: Raman spectroscopy and autofluorescence imaging, detecting relevant bladder cancer biomarkers. Once the models have revealed which biomarkers to look for, fresh ex vivo tumour biopsies from patients will be tested, ensuring clinical translation of results.
Finally, using advanced AI supervised learning techniques, images of patient tumours will be analysed to identify tumour features relevant for diagnosis. By doing so, Amplitude can develop automated classification algorithms for tumour discrimination, improving bladder cancer diagnosis further still.