Wide-field imaging of fluorescent deoxy-glucose in ex vivo malignant and normal breast tissue.

Rapid in situ determination of surgical resection margins during breast cancer surgery would reduce patient time under anesthesia. We present preliminary data supporting the use of a fluorescent glucose analog (2-NBDG) as an optical contrast agent to differentiate freshly excised breast tissue containing cancerous cells from normal breast tissue. Multi-spectral images of 14 breast cancer specimens acquired before and after incubation with 2-NBDG demonstrated increased fluorescent signal in all of the malignant tissue due to increased 2-NBDG consumption. We demonstrate that 2-NBDG has potential as an optical contrast agent to differentiate cancerous from non-cancerous tissue.

Influence of illumination-collection geometry on fluorescence spectroscopy in multilayer tissue.

Device-tissue interface geometry influences both the intensity of detected fluorescence and the extent of tissue sampled. Previous modelling studies have often investigated fluorescent light propagation using generalised tissue and illumination-collection geometries. However, the implementation of approaches that incorporate a greater degree of realism may provide more accurate estimates of light propagation. In this study, Monte Carlo modelling was performed to predict how illumination-collection parameters affect signal detection in multilayer tissue. Using the geometry and optical properties of normal and atherosclerotic aortas, results for realistic probe designs and a semi-infinite source-detection scheme were generated and compared. As illumination-collection parameters, including single-fibre probe diameter and fibre separation distance in multifibre probes, were varied, the signal origin deviated significantly from that predicted using the semi-infinite geometry, The semi-infinite case under-predicted the fraction of fluorescence originating from the superficial layer by up to 23% for a 0.2mm diameter single-fibre probe and over-predicted by 10% for a multifibre probe. These results demonstrate the importance of specifying realistic illumination-collection parameters in theoretical studies and indicate that targeting of specific tissue regions may be achievable through customisation of the illumination-collection interface. The device- and tissue-specific approach presented has the potential to facilitate the optimisation of minimally invasive optical systems for a wide variety of applications.

Understanding the contributions of NADH and collagen to cervical tissue fluorescence spectra: modeling, measurements, and implications.

OBJECTIVE: At 380 nm excitation, cervical tissue fluorescence spectra demonstrate characteristic changes with both patient age and the presence of dysplasia. A Monte Carlo model was developed in order to quantitatively examine how intrinsic NADH and collagen fluorescence, in combination with tissue scattering and absorption properties, yield measured tissue spectra. METHODS: Excitation-emission matrices were measured for live cervical cells and collagen gel phantoms. Fluorescence microscopy of fresh tissue sections was performed to obtain the location and density of fluorophores as a function of patient age and the presence of dysplasia. A Monte Carlo model was developed which incorporated measurements of fluorophore line shapes and spatial distributions. RESULTS: Modeled spectra were consistent with clinical measurements and indicate that an increase in NADH fluorescence and decrease in collagen fluorescence create clinically observed differences between normal and dysplastic tissue spectra. Model predictions were most sensitive to patient age and epithelial thickness. CONCLUSIONS: Monte Carlo techniques provide an important means to investigate the combined contributions of multiple fluorophores to measured emission spectra. The approach will prove increasingly valuable as a more sophisticated understanding of in vivo optical properties is developed.

Autofluorescence microscopy of fresh cervical-tissue sections reveals alterations in tissue biochemistry with dysplasia.

Fluorescence spectroscopy offers an effective, noninvasive approach to the detection of precancers in multiple organ sites. Clinical studies have demonstrated that fluorescence spectroscopy can provide highly sensitive, specific and cost-effective diagnosis of cervical precancers. However, the underlying biochemical mechanisms responsible for differences in the fluorescence spectra of normal and dysplastic tissue are not fully understood. We designed a study to assess the differences in autofluorescence of normal and dysplastic cervical tissue. Transverse, fresh tissue sections were prepared from colposcopically normal and abnormal biopsies in a 34-patient study. Autofluorescence images were acquired at 380 and 460 nm excitation. Results showed statistically significant increases in epithelial fluorescence intensity (arbitrary units) at 380 nm excitation in dysplastic tissue (106 +/- 39) relative to normal tissue (85 +/- 30). The fluorophore responsible for this increase is possibly reduced nicotinamide adenine dinucleotide. Stromal fluorescence intensities in the dysplastic samples decreased at both 380 nm (102 +/- 34 [dysplasia] vs 151 +/- 44 [normal]) and 460 nm excitation (93 +/- 35 [dysplasia] vs 137 +/- 49 [normal]), wavelengths at which collagen is excited. Decreased redox ratio (17-40% reduction) in dysplastic tissue sections, indicative of increased metabolic activity, was observed in one-third of the paired samples. These results provide valuable insight into the biological basis of the differences in fluorescence of normal and precancerous cervical tissue.

Laser scanning confocal microscopy of cervical tissue before and after application of acetic acid.

OBJECTIVES: The use of high-resolution in vivo confocal imaging may offer a clinically useful adjunct to standard methods for the diagnosis and screening of epithelial precancers. This study assesses the feasibility of real-time confocal reflectance imaging of cervical tissue and the use of acetic acid as a contrast agent to increase visualization of cell nuclei. STUDY DESIGN: A confocal microscope was used to image cervical cells and colposcopically normal and abnormal cervical biopsy specimens. Images were obtained before and after the application of 6% acetic acid. RESULTS: The confocal imaging system resolved subcellular detail throughout the entire epithelial thickness. Normal and abnormal tissues were clearly able to be differentiated. Addition of acetic acid enhanced nuclear signal in all acquired images. CONCLUSION: High-contrast reflected light images of cervical tissue are attainable in near real time. Acetic acid significantly increases light scattering from cell nuclei, which may partially explain why acetowhitening occurs.

A pulsed finite-difference time-domain (FDTD) method for calculating light scattering from biological cells over broad wavelength ranges.

We combine the finite-difference time-domain method with pulse response techniques in order to calculate the light scattering properties of biological cells over a range of wavelengths simultaneously. The method we describe can be used to compute the scattering patterns of cells containing multiple heterogeneous organelles, providing greater geometric flexibility than Mie theory solutions. Using a desktop computer, we calculate the scattering patterns for common homogeneous models of biological cells and also for more complex representations of cellular morphology. We find that the geometry chosen significantly impacts scattering properties, emphasizing the need for careful consideration of appropriate theoretical models of cellular scattering and for accurate microscopic determination of optical properties.

Optimal fluorescence excitation wavelengths for detection of squamous intra-epithelial neoplasia: results from an animal model.

Using the hamster cheek pouch carcinogenesis model, we explore which fluorescence excitation wavelengths are useful for the detection of neoplasia. 42 hamsters were treated with DMBA to induce carcinogenesis, and 20 control animals were treated only with mineral oil. Fluorescence excitation emission matrices were measured from the cheek pouches of the hamsters weekly. Results showed increased fluorescence near 350-370 nm and 410 nm excitation and decreased fluorescence near 450-470 nm excitation with neoplasia. The optimal diagnostic excitation wavelengths identified using this model - 350-370 nm excitation and 400-450 nm excitation - are similar to those identified for detection of human oral cavity neoplasia.

Light scattering from cells: finite-difference time-domain simulations and goniometric measurements.

We have examined the light-scattering properties of inhomogeneous biological cells through a combination of theoretical simulations and goniometric measurements. A finite-difference time-domain (FDTD) technique was used to compute intensity as a function of scattering angle for cells containing multiple organelles and spatially varying index of refraction profiles. An automated goniometer was constructed to measure the scattering properties of dilute cell suspensions. Measurements compared favorably with FDTD predictions. FDTD and experimental results indicate that scattering properties are strongly influenced by cellular biochemical and morphological structure.

Reflectance spectroscopy with polarized light: is it sensitive to cellular and nuclear morphology.

We present a method for selective detection of size-dependent scattering characteristics of epithelial cells in vivo based on polarized illumination and polarization sensitive detection of scattered light. We illustrate the method using phantoms designed to simulate squamous epithelial tissue and progressing to epithelial tissue in vitro and in vivo. Elastic light scattering spectroscopy with polarized illumination/detection dramatically reduces background signals due to both diffuse stromal scattering and hemoglobin absorption. Resulting spectra can be described as a linear combination of forward and backscattering components determined from Mie theory. Nuclear sizes and refractive indices extracted by fitting experimental spectra to this model agree well with previous measurements. Reflectance spectroscopy with polarized light can provide quantitative morphological information which could potentially be used for non-invasive detection of neoplastic changes.

Near real time confocal microscopy of cultured amelanotic cells: sources of signal, contrast agents and limits of contrast.

The use of high resolution, in vivo confocal imaging for noninvasive assessment of tissue pathology may offer a clinically important adjunct to standard histopathological techniques. To augment the present understanding of both the capabilities and limitations of in vivo confocal imaging, we investigated cellular sources of image contrast in amelanotic tissues, how contrast can be enhanced with external agents and how contrast is degraded by the scattering of overlying cells. A high-resolution reflected light confocal microscope was constructed and used to obtain images of various types of unstained amelanotic cells in suspension in real time before and after the addition of contrast agents. Reflectance images were compared to phase contrast images and electron micrographs to identify morphology visible with real time reflected light confocal microscopy. Mechanisms which decrease image contrast, including interference effects and scattering in overlying layers of cells, were considered. In amelanotic epithelial cells, fluctuations in the nuclear index of refraction provide signal which can be imaged even under several overlying cell layers. Acetic acid is an external contrast agent which can enhance this nuclear backscattering. Image contrast is degraded by the presence of multiple scattering in overlying cell layers. The degradation of image contrast by cell scattering depends on the scattering phase function; in vitro models which use polystyrene microspheres to approximate tissue underestimate the actual degradation caused by cell scattering. The loss in contrast can be explained using a finite difference time domain model of cellular scattering. We conclude that near real time reflected light confocal microscopy can be used to study cell morphology in vivo. Contrast degradation due to overlying tissue is a concern and cannot adequately be modeled using conventional tissue phantoms; however, acetic acid may be used to substantially increase intrinsic contrast, allowing imaging at significant depths despite distortion from overlying layers. © 1998 Society of Photo-Optical Instrumentation Engineers.