Development of Minimally-Invasive Optical Methods to Individualize the Doses Used for Therapeutic Applications of Light

Despite the experience gained over several decades in various types of light-based medical treatments, the optimization of the corresponding therapeutic protocols and accurate forecasting of their outcome have not yet been achieved in many cases. The difficulty often arises from the heterogeneity of living tissues, their variable optical properties, and from the heterogeneous distribution of the photoactive or photosensitive substances – whether naturally present in the tissue or artificially adde. Our work focuses on the individualization and control of irradiation parameters, in order for the physician to be able to elicit a predictable clinical response in the irradiated tissues. In this thesis, we present three separate studies in which we tried to evaluate the possibility of individualizing and optimizing the corresponding clinical outcomes by measuring or monitoring certain, particular parameters. In a first clinical study, performed at the medical practice of Dr Vezzola, MD, in Saló, Italy, the human eye's retinal reflectance was measured and mapped, in the framework of subthreshold thermal laser therapy, using an excitation wavelength identical to that of the treatment laser, i.e. at 810 nm. The specific goal of this study was to correlate the occurrence of retinal burns with the measured infrared retinal reflectance. This study was performed using a modified fundus camera to record infrared reflectance images of the retina, and by recording the slit-lamp based laser therapy parameters (irradiation parameters and spot location) in such a way so as to overlay the map of the laser treatment spots on the corresponding reflectance fundus image. The clinical study demonstrated the expected existence of spatial variations in light reflectance at 810 nm (probably due to changes of the tissue absorption), which we then tried to relate to the occurrences of retinal burns observed during the laser treatment. The analysis of the results obtained with the applied conditions, did not however show a clear correlation between the local retina reflectance, the laser beam parameters, and the occurrence of retinal burns. Therefore we postulate that either the absorbing structures of the retina cannot be seen with the imaging device we used, possibly due to its limited resolution, or other important elements play a role in the laser-tissue interactions during this type of lasers-light interaction with the local retinal tissue. The second study was pre-clinical, performed at EPFL, and aimed at monitoring in real-time the tissular oxygen concentration during photodynamic therapy (PDT). It was performed in vivo on the chicken embryo's chorio-allantoic membrane (CAM) model, which was submitted to aminolevulinic acid (ALA)-based PDT. The molecular oxygen, which is thought to be an essential actor in the cascade of reactions leading to the tissular PDT effect, is actually the main molecule responsible for the photosensitizer's (PS) triplet state quenching. Therefore, the delayed fluorescence lifetime of the photosensitizer, protoporphyrin IX (PpIX), was measured with a specially designed and assembled, optical fiber-based, time-resolved spectrofluorometer, and used as a proxy for tissular pO2. Simultaneously, vascular damages caused by PDT were characterized and quantified, to check for correlation between the two parameters. Using the PS's delayed fluorescence lifetime to evaluate tissular pO2 proved to be a quite reasonable strategy, due to the fact that it is possible to measure the pO2 at the location of the PS molecule. The study's results demonstrate a robust, linear correlation between tissular pO2 reduction and vascular damage extent. They also suggest that the amount of oxygen consumed during PDT could be a useful, measurable parameter for assessing and/or controlling the PDT's therapeutic effect. The third, clinical, study aimed at measuring the fluorescence photobleaching of the PpIX photosensitizer due to PDT. This study was performed at the "Hôpitaux Universitaires de Genève – HUG", in Geneva, Switzerland, in collaboration with Dr Denis Salomon, MD, and Dr Behrooz Kasraee, MD, in the framework of a series of standard clinical PDT treatments of aktinic keratoses (AK), which is a pre-cancerous skin lesion. The lesions' fluorescence intensity was quantified with a specially adapted quantitative imaging device, using a homogeneous and constant intensity fluorescence excitation light. The specific goal was to check for a relation between the extent of photobleaching of the PS (in this case PpIX, which was induced by the administration of Metvix®) and the clinical outcome (disappearance of AK) evaluated several months after the treatment. The study's results show that the amount of photobleached PS is strongly and linearly correlated to the fluorescence measured before the treatment. Likewise, the preliminary assessment of the clinical outcomes confirms the existence of a correlation of these outcomes with the PS's fluorescence bleaching, thus making it possible, in principle, to select and optimize the PDT's irradiation parameters before starting the treatment. The results of this study demonstrate the relevance of measuring the PS's photo-bleaching for optimizing PDT and forecasting of its outcome.

Single molecule detection and fluorescence correlation spectroscopy on surfaces

Kai Hassler

In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

EPFL2006

Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

André Studzinski, Hubert van den Bergh, Georges Wagnières

We describe a new fluorescence imaging device for clin. cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochem. properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radiofrequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is sepd. in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calc. in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.

Springer US1997

Endoscopic Fluorescence Imaging

Blaise Lovisa

Since several decades, the physicians are able to access hollow organs with endoscopic methods, which serve both as diagnostic and surgical means in a wide range of disciplines of the modern medicine (e.g. urology, pneumology, gastroenterology). Unfortunately, white light (WL) endoscopy displays a limited sensitivity to early pre-cancerous lesions. Hence, several endoscopic methods based on fluorescence imaging have been developed to overcome this limitation. Both endogenous and exogenously-induced fluorescence have been investigated, leading to commercial products. Indeed, autofluorescence bronchoscopy, as well as porphyrin-based fluorescence cystoscopy, are now on the market. As a matter of fact, fluorescence-based endoscopic detection methods show very high sensitivity to pre-cancerous lesions, which are often overlooked in WL endoscopy, but they still lack specificity mainly due to the high false-positive rate. Although most of these false positives can easily be rejected under WL observation, tissue abnormalities such as inflammations, hyperplasia, and metaplasia are more difficult to identify, often resulting in supplementary biopsies. Therefore, the purpose of this thesis is to study novel, fast, and convenient method to characterize fluorescence positive spots in situ during fluorescence endoscopy and, more generally, to optimize the existing endoscopic setup. In this thesis, several clinical evaluations were conducted either in the tracheo-bronchial tree and the urinary bladder. In the urinary bladder, fluorescence imaging for detection of non-muscle invasive bladder cancer is based on the selective production and accumulation of fluorescing porphyrins, mainly protoporphyrin IX (PpIX), in cancerous tissues after the instillation of Hexvix® during one hour. In this thesis, we adapted a rigid cystoscope to perform high magnification (HM) cystoscopy in order to discriminate false from true fluorescence positive findings. Both white light and fluorescence modes are possible with the magnification cystoscope, allowing observation of the bladder wall with magnification ranging between 30× – for standard observation – and 650×. The optical zooming setup allows adjusting the magnification continuously in situ. In the high magnification regime, the smallest diameter of the field of view is 600 microns and the resolution is 2.5 microns, when in contact with the bladder wall. With this HM cystoscope, we characterized the superficial vascularization of the fluorescing sites in WL (370–700 nm) reflectance imaging in order to discriminate cancerous from non-cancerous tissues. This procedure allowed us to establish a classification based on observed vascular patterns. 72 patients subject to Hexvix® f luorescence cystoscopy were included in the study. Comparison of HM cystoscopy classification with histopathology results confirmed 32/33 (97%) cancerous biopsies, and rejected 17/20 (85%) non-cancerous lesions. No vascular alteration could be observed on the only positive lesion that was negative in HM mode, probably because this sarcomatoid carcinoma was not originating in the bladder mucosa. We established with this study that a magnification ranging between 80× and 100× is an optimal tradeoff to perform both macroscopic PDD and HM reflectance imaging. In order to make this approach more quantitative, different algorithms of image processing (vessel segmentation and skeletonisation, global information extraction) were also implemented in this thesis. In order to better visualize the vessels, we improved their contrast with respect to the background. Since hemoglobin is a very strong absorber, we targeted the two hemoglobin absorption peaks by placing appropriate bandpass filters (blue 405±50 nm, green 550±50 nm) in the light source. HM cystoscopy was then performed sequentially with WL, blue and green illumination. The two latter showed higher vessel-to-background contrast, identifying different layers of vascularization due to the light penetration depth. During fluorescence cystoscopy, we often observed that the images are somehow "blurred" by a greenish screen between endoscope tip and bladder mucosa. Since this effect is enhanced by the urine production, it is more visible with flexible scopes (lower flushing capabilities) and imaging systems that collect only autofluorescence as background. Indeed, when the bladder is not flushed regularly, greenish flows coming out of the ureters can easily be observed. For this reason, it is supposed that some fluorophores contained in the urine are excited by the photodetection excitation light, and appear greenish on the screen. This effect may impair the visualization of the bladder mucosa, and thus cancerous lesions, and lowers sensitivity of the fluorescence cystoscopy. In this thesis, we identified the main metabolites responsible for the liquid fluorescence, and optimized the spectral design accordingly. In the tracheo-bronchial tree, the fluorescence contrast is based on the sharp autofluorescence (AF) decrease on early cancerous lesions in the green spectral region (around 500 nm) and a relatively less important decrease in the red spectral region (> 600 nm) when excited with blue-violet light (around 410 nm). It has been shown over the last years, that this contrast may be attributed to a combined effect of epithelium thickening and higher concentration of hemoglobin in the tissues underneath the (pre-)cancerous lesions. In this thesis, we contributed to the definition of the input design of several new prototypes, that were subsequently tested in the clinical environment. We first showed that narrow-band excitation in the blue-violet could increase the tumor-to-normal spectral contrast in the green spectral region. Then, we quantified the intra- and inter-patient variations in the AF intensities in order to optimize the spectral response of the endoscopic fluorescence imaging system. For this purpose, we developed an endoscopic reference to be placed close to the bronchial mucosa during bronchoscopy. Finally, we evaluated a novel AF bronchoscope with blue-backscattered light on 144 patients. This new device showed increased sensitivity for pre-neoplastic lesions. Similar to what we observed in the bladder, it is likely that developing new imaging capabilities (including vascular imaging) will facilitate discriminating true from false positive in AF bronchoscopy. Here, we demonstrated that this magnification allowed us to resolve vessels with a diameter of about 30 µm. This resolution is likely to be sufficient to identify Shibuya's vascular criteria (loops, meshes, dotted vessels) on AF positive lesions. This criteria allow him to recognize pre-cancerous lesions, and thus can potentially decrease the false-positive rate with our AF imaging system. This magnification was also showed to be better for routine bronchoscopy, since it delivers sharper and more structured images to the operator.