Mieke Bus

42 Chapter 3 resolution is achieved using lasers and optics, which are combined with a very small hole (pin-hole) that acts as a diaphragm in the microscope objective. This pinhole ensures that only light from the focus in the tissue is collected. The light that is out of focus is rejected by the pin-hole (Figure 6). CLE uses fluorescence from fluorescein to stain tissue micro architec- ture and small vessels, which rapidly diffuses through the body after intravenous injection. Recent advantages in instrument miniaturization have led to the development of flexible, fiberoptic confocal microscopes that can be used with standard endoscopy to provide real-time information on tumour grade. In an ex-vivo study urothelial cells and lamina pro- pria were clearly recognized. (22) Drawback of CLE is sensitivity to tissue movement leading to motion artefacts that could result in blurred images. Although commercially available CLE devices are now available with decreased probe size with a wider field of view and an improved imaging of the microarchitecture of tissue, it also results in a lower cellular resolu- tion, which is needed for grading. (23) Techniques based on light scattering Optical Coherence Tomography Optical Coherence Tomography (OCT) is analogous to ultrasonography, using back-scat- tered light instead of back-reflected sound waves to produce micrometre-scale resolution, cross-sectional images (Table 1). (24) Recent OCT research investigates this technology in the diagnostic workup of several epithelial cancers. (25-27) In OCT, layered tissue anatomy can be distinguished. Interruption or absence of layered tissue under a visible lesion is indicative for tumour stage. However, light scattering causes a decrease of OCT signal magnitude over depth, and limits the imaging range to approximately 2mm depth. The rate of OCT signal decrease with depth is quantified by the attenuation coefficient (µ oct ) that allows in-vivo differentiation between different tissue types (Figure 7). (26-29) This distinction results from differences in intra- and extracellular organization of the tissue, which is reflected in the light scattering properties. Measurement of µ oct is therefore sensitive to the differences in organi- zation associated with different grades of the lesion. The combination of real-time, high-res- olution images and extraction of the optical attenuation coefficient gives OCT the ability to provide real-time information on tumour stage and grade in the upper urinary tract. The potential of OCT for staging/diagnosis of UTUC has been investigated ex-vivo in the porcine and human ureter where it clearly distinguished the ureteral wall layers, particularly the urothelium and lamina propria. (30-32) When compared to endoluminal ultrasonography, OCT can significantly better distinguish the wall layers of ex-vivo porcine ureter. (33) An in-vivo human pilot study in the ureter showed that normal appearing urothelium, the layered tissue anatomy including basement membrane, CIS and visible protrusions can be visual- ized on OCT images. Towards grading, UUT-OCT was able to visually differentiate between