Although fixed-excitation laser-induced fluorescence (LIF) has been shown to be successful for differentiating normal from malignant tissues, this technique does not distinguish low-grade dysphasia from Barrett's mucosa without dysplasia. A limitation of the conventional fixed-excitation LIF technique results because it does not often provide the spectral specificity needed to provide clear “spectral fingerprints” of normal and dysplastic tissues mainly because the flourescence of tissue is a composite of fluorescence emissions from various tissue components. For example, tissue components generally include endogenous fluorophors such as aromatic amino acids (e.g., tryptophan, tyrosine, phenylalanine), structural matrix proteins (e.g., collagen, elastin), nicotiamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), porphyrins, Lipo-pigments (e.g., ceroids, lipofuscin), and other biological components. As a result, applied to tissue diagnostics LIF provides spectra that are poorly resolved and featureless because of spectral overlap between the emissions from individual tissue components. Thus, the laser used in LIF can only improve the sensitivity but cannot enhance the specificity (selectivity).
In addition to spectral specificity problems, current instrumentation for cancer and other tissue diagnostics have other serious limitations. One major limitation of the conventional fluorescence technique results from the use of a fixed-wavelength excitation source (e.g., a monochromatic laser). As noted above, each component in tissue (e.g., collagen, FAD, porphyrins, NADH, etc.) has specific absorption and emission spectra occurring in particular spectral ranges. When a single fixed laser line is used, it is difficult, if not impossible, to excite all the biological components under optimal conditions.
Synchronous luminescence (SL) methodology is an improved technology over LIF and provides a way to measure the luminescence signal and spectral fingerprints for rapid screening of complex chemical samples. The general theory of the SL method has been described previously in “Synchronous Excitation Spectroscopy,” authored by the inventor of the present application T. Vo-Dinh, in Modern Fluorescence Spectroscopy, Chapter 5, Ed. by E. L. Wehry (Plenum Publ. Corp. 1981), which is incorporated herein by reference in its entirety. In contrast to SL, conventional luminescence spectroscopy uses either a fixed-wavelength excitation (λex) to produce an emission spectrum or a fixed wavelength emission (λem) to record an excitation spectrum. With SL, the luminescence signal is recorded while both λem and λex are simultaneously scanned. A constant wavelength interval is generally maintained between the excitation and the emission monochromators throughout the spectrum. As a result, the observed intensity Is of the synchronous signal can be written as a product of two functions as follows:Is(λex, λem)=k c Ex(λex)·EM(λex)  (1)where:    k=a constant,    c=concentration of the analyte,    Ex=excitation function, and    EM=emission function.
For a single molecular species the observed intensity Is is simplified often to a single peak, and the bandwidth is narrower than for the conventional emission spectrum. Since the SL spectrum of each component becomes sharper due to the band-narrowing effect of the SL technique, the resulting fluorescence spectrum of the tissues sample becomes better resolved with a plurality of readily identifiable sharp individual emission peaks.
In many medical applications (e.g., endoscopies), the use of optical fibers is required in order to perform in situ and in vivo measurements in an imaging mode. The inventor has previously disclosed several technologies based on fluorescence and synchronous luminescence for medical applications, such as U.S. Pat. No. 5,599,717 entitled “Advanced Synchronous Luminescence System for Medical Diagnostics,” and U.S. Pat. No. 5,938,617 ('617) entitled “Advanced Synchronous Luminescence System for the Detection of Biological Agents and Infectious Pathogens”.
'617 teaches a SL system 100 which is shown schematically in FIG. 1. The system 100 includes a laser 102 which provides outputs a light beam 104 having a given wavelength. The light beam 104 is coupled to a structure 106 for changing its wavelength, such as a multi-dye module (MDM) 106. The output of the MDM 106 is delivered, through a focusing lens 108, to a first optical fiber or bundle of fibers 110 for transmitting the excitation radiation to the sample 122 being investigated.
The optical fiber 110 transmits the excitation radiation beam to a probe 120 which is juxtaposed to sample 122. A second optical fiber or bundle of fibers 125 transmits the fluorescence emission from sample 122 to a detector 130. The detector 130 comprises a monochromator (MON) 131 and a photomultiplier (PM) 132. A boxcar integrator (BCI) 134, synchronized with the laser pulse via a pulse generator (PG) 136 acting as a trigger is used to record and process the emitted fluorescence signal. A synchronous scanning device (SS) 138 ensures that the excitation radiation (λex) and the emission radiation (λem) are maintained at a constant interval. A portable computer 140, or other suitable data collection, analysis and/or display devices, can be used to generate a synchronous luminescence spectra which can be compared to spectra from known healthy tissue samples to detect tissue anomalies.
Significantly, system 100 includes a bifurcated fiber probe arrangement where a first probe 110 provides excitation radiation to sample 122 in a first location while a second probe 125 receives emitted radiation from sample 122 from a second sample location. Thus, the excitation and detection locations are different. As a result, co-registration of the excitation and emission is not possible. System 100 also does not generally allow precise determination of the emission location as emissions can originate from a plurality of locations around the excitation location.