Quantitative Spectroscopic Imaging: Clinical instrumentation

Motivation

Early detection is essential for managing cancer, since treatment is much more successful when lesions are diagnosed at an early, noninvasive stage. Current cancer diagnosis often employs visual inspection of a wide area of tissue followed by biopsy of suspicious sites. This practice is problematic for two reasons: (1) early cancers are not always detectable by visual inspection, so, unavoidably, unnecessary biopsies are taken for precautionary reasons and invisible lesions are missed; and (2) biopsy suffers from undersampling, and the results are subjective and the resulting pathology can be subject to low inter-observer agreement. Furthermore, biopsy results often are not available immediately, resulting in delayed treatment and patient anxiety.

Much attention has been focused on spectroscopy, particularly reflectance and fluorescence spectroscopy, to overcome these problems. Reflectance and fluorescence are known to exhibit spectral features associated with the different morphology and biochemistry of normal and cancerous tissues. These techniques have the capability to detect invisible lesions, to provide quantitative diagnostic information for objective evaluation, and to be implemented for wide-area imaging, essential requirements for most applications. Wide-area imaging of spectroscopic information is important for complete acquisition of data over a large field of tissue, and to overcome the under-sampling problem associated with biopsy.

Contact probe spectroscopy

Studies of reflectance and fluorescence for tissue diagnosis using optical fiber contact probes for light delivery and collection have been performed by numerous groups in the cervix, oral cavity, esophagus, colon, lung, and bladder, with various degrees of quantitative analysis. Our own work has focused on developing quantitative methods for tissue diagnosis.  We use diffuse reflectance and fluorescence, in combination, to extract quantitative information about morphological and biochemical tissue constituents. We call the method quantitative spectroscopy (QS). Diffuse reflectance spectra from tissue are analyzed using a well-developed model to obtain information about hemoglobin concentration and saturation, light scattering parameters, and other tissue characteristics.  This method is known as diffuse reflectance spectroscopy (DRS). Tissue fluorescence, collected from the same spot at the same time, is analyzed using the diffusely reflected light to remove spectral distortions, resulting in the “intrinsic fluorescence” that would be observed in the absence of scattering and absorption, from which contributions from tissue fluorophores can then be extracted. This method is known as intrinsic fluorescence spectroscopy (IFS). Histological parameters are then extracted by fitting the observed spectra to parameters such as tissue density, blood concentration and oxygenation, and concentrations of collagen and reduced nicotinamide adenine dinucleotide (NADH), determined from calibration of physical models of tissue with known features.

Contact probe techniques are promising, but, like biopsy, suffer from undersampling. To overcome this, various groups have been developing techniques to incorporate wide area imaging in fluorescence and reflectance tissue diagnosis, using a variety of approaches with different degrees of quantitative analysis [22-25]. Recently, our laboratory has extended our model-based, quantitative approach to wide field imaging. We call the new modality quantitative spectroscopic imaging (QSI). Data are collected by means of a non-contact “virtual” probe, imaged at the tissue surface. This virtual probe is then raster scanned to interrogate a wide tissue area (~4cm2), one spot (1mm2) at a time. The quantitative measurements of tissue properties enable the spectra for each pixel to be analyzed using our contact probe methodology. Hence, the QSI images are directly interpretable in terms of histological features, thus providing a robust diagnosis.

Instrumentation

Studies of reflectance and fluorescence for tissue diagnosis using optical fiber contact probes for light delivery and collection have been performed by numerous groups in the cervix, oral cavity, esophagus, colon, lung, and bladder, with various degrees of quantitative analysis. Our own work has focused on developing quantitative methods for tissue diagnosis.  We use diffuse reflectance and fluorescence, in combination, to extract quantitative information about morphological and biochemical tissue constituents. We call the method quantitative spectroscopy (QS). Diffuse reflectance spectra from tissue are analyzed using a well-developed model to obtain information about hemoglobin concentration and saturation, light scattering parameters, and other tissue characteristics.  This method is known as diffuse reflectance spectroscopy (DRS). Tissue fluorescence, collected from the same spot at the same time, is analyzed using the diffusely reflected light to remove spectral distortions, resulting in the “intrinsic fluorescence” that would be observed in the absence of scattering and absorption, from which contributions from tissue fluorophores can then be extracted. This method is known as intrinsic fluorescence spectroscopy (IFS). Histological parameters are then extracted by fitting the observed spectra to parameters such as tissue density, blood concentration and oxygenation, and concentrations of collagen and reduced nicotinamide adenine dinucleotide (NADH), determined from calibration of physical models of tissue with known features.

Figure 1.(a) QSI instrumentation principle. Green beams are illumination and red beams are collection. Optics delivers a spot of light to the tissue surface. The spot is similar in diameter to that of an optical fiber contact probe. The area from which reflectance and fluorescence are collected is also similar. (b) Block diagram of the QSI instrument. (c) Schematic diagram of the optical head.

We note that the characteristics of our non-contact probe (spot size ~ 1mm in diameter and NA ~ 0.02) differ somewhat from that of our contact probe studies (spot size ~ 0.8mm in diameter and NA ~ 0.22). As discussed below, the probe parameters are incorporated in our reflectance and fluorescence models, and differences in probe geometries can be taken into account, so the correct tissue parameters can be obtained from measurements with either probe. This robustness is an important virtue of our quantitative approach.

The QSI instrument has been developed for clinical settings, and must be portable. As shown in Fig. 1(b), portability is achieved by placing the heaviest components in a mobile cart and creating a separate lightweight, mobile optical head to be operated by the physician. The design is modular. The three larger modules, mounted in a cart, are connected to the optical head by means of optical fibers. Module 1, the light source module, consists of a 75W CW white light arc lamp (Simplicity series, Newport Corp.) and a nitrogen laser that delivers 337nm light pulses of duration <3.5 ns and energy 175 μJ at 20 Hz (NL100, Stanford Research Systems Inc.). Module 2 contains a spectrograph/CCD unit (Princeton Instruments Corp.). Module 3 is the computer, which controls data acquisition and analyzes the data. National Instruments Labview software and DAQ data acquisition hardware are used to control and coordinate the various components. Module 4, is mounted on an articulated arm for easy maneuvering. It is located on a smaller cart with wheels, so that the larger equipment cart can be located away from the patient, since space in hospital procedure rooms is limited.

Fig. 1(c) is a schematic diagram of the optical head. White light (with NA ~ 0.02) from the xenon arc lamp (CW) illuminates a 1mm diameter “diagnostic spot” on the tissue about 20cm away from the optical head. Diffusely reflected light from a 2mm diameter circle centered on the diagnostic spot is relayed back to the optical head and focused onto eight collection fibers coupled to a spectrograph and CCD. Each of the eight fibers collects light returning from the tissue in different directions, which helps to avoid specular reflection. Next, 337nm light (with NA ~ 0.02) from the nitrogen laser illuminates the same diagnostic spot, and fluorescence is collected in the same manner as diffuse reflectance. These two measurements are equivalent to the reflectance and fluorescence measurements of a contact probe.

Wide area coverage is achieved by means of a 2D scanning mirror (OIM102, Optics in Motion LLC), which can tilt by up to ± 1.5° along two orthogonal axes, and thus raster scan the diagnostic spot across a 2.1cm x 2.1cm region of the tissue surface in a stepwise fashion. At each mirror position, a reflectance measurement is made, followed immediately by a fluorescence measurement. Each pair of measurements is associated with a tissue location which correlates with the mirror position. However, this correlation can be affected by patient movement during the procedure. Therefore, the patient’s movement needs to be tracked in order to take any shift in tissue position into account. An onboard color video camera (QICAM, QImaging Corp.) tracks the patient movement relative to the instrument by acquiring one photograph of the tissue every second during the procedure. Two white light LEDs (CCS Inc., not shown), which provide extra illumination for the video camera, are turned off during reflectance and fluorescence measurements. The total time required for a 2.1cm x 2.1cm scan is approximately 90s at present, but this time can be considerably reduced by using a CCD camera (for spectra) with the on-board memory (to eliminate the data transfer time and greatly reduce the data writing time); and increasing the angle of optical collection (to decrease the CCD exposure time).

Recent Publications

1. Chung-Chieh Yu, Condon Lau, Geoffrey O'Donoghue, Jelena Mirkovic, Sasha McGee, Luis Galindo, Alphi Elackattu, Elizabeth Stier, Gregory Grillone, Kamran Badizadegan, Ramachandra R. Dasari, and Michael S. Feld, “Quantitative spectroscopic imaging for non-invasive early cancer detection,” Optics Express Vol. 16, Iss. 20, pp. 16227–16239 (2008).