Miniaturisation and testing of an optical interference block for fluorescence imaging in capsule endoscopy

Early detection of gastrointestinal cancer is crucial to increase the life span of patients. The implementation of new imaging modalities, such as fluorescence imaging, in traditional endoscopy is the key in the detection of early signs of cancer. Fluorescence imaging techniques for clinical applications can be divided in two groups defined as autofluorescence imaging and fluorescence-labelling imaging. The former exploits the natural green fluorescence emitted by human tissues when excited by blue or ultra-violet light. Detection of cancer through autofluorescence imaging relies on the fact that cancer tissues have a much lower autofluorescence signal than healthy tissues. On the other hand, fluorescence-labelling imaging is used when the difference in autofluorescence between cancer and healthy surroundings is too weak to detect. Therefore, external fluorescence agents are used to target and label cancer lesions. Although traditional endoscopy has been successfully equipped with fluorescence imaging capabilities, the discomfort caused in patients and the incapability to reach the small intestine represent two main limitations. Fluorescence capsule endoscopy can enhance diagnostic accuracy with less inconvenience for patients. The optical components in traditional endoscopes are bulky and implemented outside the body of the patients. Therefore, there is a demand to develop highly miniaturised optical components for integration in capsule endoscopy. This thesis describes the design, fabrication, characterisation, and testing of a 5 mm x 6 mm x 6 mm optical interference block with the capability of fluorescence imaging in capsule endoscopy. The block accommodates ultrathin filters for optical isolation and was successfully integrated with a sensitive 64 x 64 pixels complementary metal oxide semiconductor single photon avalanche diode array to detect green fluorescence from Flavin Adenine Dinucleotide. This coenzyme is among the fluorophores responsible for autofluorescence in human tissues. The fluorescence-labelling capabilities of the imaging system were also tested to detect fluorescence from the cancer selective molecular probe ProteoGREEN-gGluTM which was used to label colorectal cancer cells. In vitro studies were also validated using a commercial ModulusTM Microplate reader. The potential use of the miniaturised block in capsule endoscopy was further demonstrated by imaging healthy and malignant resected human tissues from the colon to detect changes in autofluorescence signal that are crucial for cancer diagnosis. The results obtained demonstrated that the system successfully imaged the differences in the autofluorescence signal from resected healthy and malignant human tissues from the colon. Moreover, results from the in vitro tests showed that the system detected changes in the fluorescence signal induced in colorectal cancer cells after labelling with ProteoGREEN-gGluTM

Capsule endoscopy compatible fluorescence imager demonstrated using bowel cancer tumours

We demonstrate a proof of concept highly miniaturised fluorescence imager and its application to detecting cancer in resected human colon cancer tissues. Fluorescence imaging modalities have already been successfully implemented in traditional endoscopy. However, the procedure still causes discomfort and requires sedation. Wireless fluorescence capsule endoscopy has the potential to improve diagnostic accuracy with less inconvenience for patients. In this paper we present a 5 mm x 6 mm x 5 mm optical block that is small enough to integrate into a capsule endoscope. The block integrates ultrathin filters for optical isolation and was successfully integrated with a sensitive CMOS SPAD array to detect green fluorescence from Flavin Adenine Dinucleotide (FAD), which is an endogenous fluorophore responsible for autofluorescence in human tissues, and fluorescence from the cancer selective molecular probe ProteoGREENTM-gGlu used to label colorectal cancer cells. In vitro studies were validated using a commercial ModulusTM Microplate reader. The potential use of the device in capsule endoscopy was further validated by imaging healthy and malignant resected human tissues from the colon to detect changes in autofluorescence signal that are crucial for cancer diagnosis

A 64x64 SPAD array for portable colorimetric sensing, fluorescence and X-ray imaging

We present the design and application of a 64x64 pixel SPAD array to portable colorimetric sensing, and fluorescence and x-ray imaging. The device was fabricated on an unmodified 180 nm CMOS process and is based on a square p+/n active junction SPAD geometry suitable for detecting green fluorescence emission. The stand-alone SPAD shows a photodetection probability greater than 60% at 5 V excess bias, with a dark count rate of less than 4 cps/µm2 and sub-ns timing jitter performance. It has a global shutter with an in-pixel 8-bit counter; four 5-bit decoders and two 64-to-1 multiplexer blocks allow the data to be read-out. The array of sensors was able to detect fluorescence from a fluorescein isothiocyanate (FITC) solution down to a concentration of 900 pM with a SNR of 9.8 dB. A colorimetric assay was performed on top of the sensor array with a limit of quantification of 3.1 µM. X-rays images, using energies ranging from 10 kVp to 100 kVp, of a lead grating mask were acquired without using a scintillation crystal

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated βTrCP, the substrate-receptor subunit of the SCFβTrCP ubiquitin ligase, and promotes βTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors

Imaging Fluorophore-Labelled Intestinal Tissue via Fluorescence Endoscope Capsule

The authors have developed a wireless fluorescence imaging capsule endoscope, potentially capable of detecting early signs of disease in the human intestine which can be missed by white-light imaging (WLI) capsule endoscopy (Figure 1). Intestinal fluorescence imaging exploits variations in tissue autofluorescence between healthy and diseased areas in response to illumination, or application of fluorescent labels which preferentially bind to diseased sites. To validate the capsule’s capability to image fluorescently-labelled tissue, a small area of a sample of ex vivo porcine small intestine was sonicated with 6 nm CdZnMg fluorescent quantum dots, and the labelled area clearly differentiated from surrounding tissue by the fluorescence imaging capsule

A 64x64 SPAD array for portable colorimetric sensing, fluorescence and x-ray imaging

Design and characterization of the designed test Single photon avalanche diode (SPAD) and the 64x64 pixel array based on the test SPAD. The test SPAD performances are compared to the state-of-the-art to prove the effectiveness of the design at low illumination condition. The 64x64 SPAD array performances are characterized and the results of three different applications (colorimetric sensing, fluorescence and x ray imaging) are reported

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated βTrCP, the substrate-receptor subunit of the SCFβTrCP ubiquitin ligase, and promotes βTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors