Progress in technology has, in recent times, had great impact in developments in the medical field. More specifically, optical methods, due to their non-invasive nature, are providing novel approaches to medical imaging, diagnosis and therapy. The motivation behind pursuing optical techniques for medical imaging is the fact that optical methods offer the advantage of achieving high-resolution image of tissue and its underlying structure using non-ionizing radiation. Several optical imaging modalities such as Optical Coherence Tomography (OCT), Time Gated Imaging, Polarization Gated Imaging and Diffuse Photon Density Wave (DPDW), have been developed in the recent past to image bio-tissue and its underlying structure with spatial resolution much higher than that can be achieved by the existing medical imaging techniques. For optical imaging, one mostly makes use of the elastically scattered (scattered without any change in frequency) light from tissue, which is the dominant component of the scattered light. It is important to note here that the scattered light also has a very weak component which is scattered in-elastically i.e. with a change in frequency via processes like fluorescence, Raman scattering etc. The inelastically scattered light is characteristic of the chemical composition and morphology of the tissue and thus can help monitor metabolic parameters of the tissue and also help discriminate diseased tissue from normal. Motivated by the fact that unlike conventional diagnostics techniques, spectroscopic measurements on human tissue may offer noninvasive, nondestructive and near real time detection of disease, both fluorescence and Raman spectroscopic approaches are being actively pursued for their diagnostic potential. Fluorescence spectroscopy has particularly been investigated for diagnosis of cancer, which is one of the most deadly diseases encountered by mankind in the past century. The ability of this technique to explore some essential features of cell metabolism has made it an attractive tool for identifying cancerous or pre-cancerous changes of tissue. Any change in the concentrations, quantum efficiency or in the binding sites and environment of fluorophores gets reflected in their fluorescence properties giving rise to a contrast in fluorescence between the normal and the cancerous tissue sites. Although, t here do appear few reports on the use of exogenous fluorophore marker for probing onset and progression of cancer, at the present stage of development, fluorescence from the endogenous tissue fluorophores (autofluorescence) had been more widely explored for cancer diagnosis. Motivated by the fact that laser induced autofluorescence spectroscopy can provide a non-invasive approach for quantitative and early diagnosis of cancer, the research interest of our group has been centered on evaluating the efficacy of the fluorescence based approach for discriminating between cancerous and normal tissues and to explore ways to improve diagnostic capability of this technique.
A major difficulty encountered in using autofluorescence from tissue for cancer diagnosis is the fact that in a turbid and multiply scattering medium like tissue, the intensity and line shape of intrinsic fluorescence from the tissue fluorophores get strongly modulated by the wavelength dependent absorption and scattering properties of tissue. This not only masks the valuable biochemical information contained in the intrinsic fluorescence, but also leads to degradation of intrinsic fluorescence contrast between cancerous and normal tissue sites. Extraction of intrinsic fluorescence by removing these distorting effects of absorption and scattering properties of tissue would thus facilitate a quantitative evaluation of the biochemical basis of the disease and may also lead to improved tissue diagnosis. We have developed two different approaches to accomplish this objective. The first approach is based on concomitant measurement of polarized fluorescence and polarized elastic scattering spectra from tissue. A photograph of the spectrofluorometer set-up used to conduct these studies is displayed in Figure 1. The polarized fluorescence normalized by the polarized elastic scattering spectra (in the wavelength range of fluorescence emission) was found to be free from the wavelength dependent modulation of absorption and scattering properties of the medium.
The underlying principle of this approach is that when excited with linearly polarized light, the polarized fraction of the detected fluorescence record fluorescence from within a few transport scattering lengths of tissue and thus is only weakly modulated by the absorption and scattering properties of tissue. Further, the propagation losses of the polarized fluorescence photons due to absorption and scattering at the excitation and the emission wavelength are similar to the propagation losses of the polarized elastically scattered photons at the same wavelength. The normalization of polarized fluorescence by the polarized elastic scattering thus serves as a means to compensate for the propagation losses and to recover the intrinsic intensity and line shape of fluorescence in a turbid medium like tissue. Since, polarized fluorescence record fluorescence signature from the superficial layer of tissue, this technique may turn out to be particularly suitable for early detection of epithelial cancer. This follows since, for epithelial cancer, the pre-cancerous changes are known to originate within the superficial epithelial layer only.
The other approach that we have developed for detection of deeply buried tumors makes use of spatially resolved fluorescence measurement from tissue. The experimental arrangement is shown in Figure 2. Typical profiles for spatially resolved fluorescence recorded from tissue are also displayed. The measured spatially resolved fluorescence from tissue is utilized for simultaneous estimation of the wavelength dependent optical transport parameters, namely, the reduced scattering coefficient (m/s), absorption coefficient (m/a), and intrinsic fluorescence spectra from tissue. The approach utilizes a hybrid diffusion theory-Monte Carlo simulation based theoretical treatment for propagation of fluorescence in tissue to estimate these parameters from the measured spatially resolved fluorescence data. While the recovered intrinsic fluorescence contains rich biochemical information on tissue, the optical transport parameters also bears useful information on morphological and physiological state of tissue.
Simultaneous determination of these parameters under the same geometric conditions and from the same probing volume of tissue might turn out to be advantageous for the development of diagnostic algorithms that could exploit both the morphological and the biochemical information contained in these parameters for optimal tissue diagnosis. Further studies are in progress in our laboratory to fully exploit the potential advantages of the developed approaches and to take laser induced fluorescence technique towards the desired goal of noninvasive and early detection of cancer.
Dr. Asima Pradhan
Department of Physics / Centre for Laser Technology,