Investigation of fiber-optic probe designs for optical spectroscopic diagnosis of epithelial pre-cancers

Abstract

Background and objectives: The first objective of this study was to evaluate the performance of fluorescence spectroscopy for diagnosing pre-cancers in stratified squamous epithelial tissues in vivo using two different probe geometries with (1) overlapping versus (2) non-overlapping illumination and collection areas on the tissue surface. Probe (1) and probe (2) are preferentially sensitive to the fluorescence originating from the tissue surface and sub-surface tissue depths, respectively. The second objective was to design a novel, angled illumination fiber-optic probe to maximally exploit the depth-dependent fluorescence properties of epithelial tissues.

Study design/materials and methods: In the first study, spectra were measured from epithelial pre-cancers and normal tissues in the hamster cheek pouch and analyzed with a non-parametric classification algorithm. In the second study, Monte Carlo modeling was used to simulate fluorescence measurements from an epithelial tissue model with the angled illumination probe.

Results: An unbiased classification algorithm based on spectra measured with probes (1) and (2), classified pre-cancerous and normal tissues with 78 and 94% accuracy, respectively. The angled illumination probe design provides the capability to detect fluorescence from a wide range of tissue depths in an epithelial tissue model.

Conclusions: The first study demonstrates that fluorescence originating from sub-surface tissue depths (probe (2)) is more diagnostic than fluorescence originating from the tissue surface (probe (1)) in the hamster cheek pouch model. However in general, it is difficult to know a priori the optimal probe geometry for pre-cancer detection in a particular epithelial tissue model. The angled illumination probe provides the capability to measure tissue fluorescence selectively from different depths within epithelial tissues, thus obviating the need to select a single optimal probe design for the fluorescence-based diagnosis of epithelial pre-cancers.

Figure 1

Flowchart outlining the steps of the multivariate statistical algorithm used for analyzing the tissue spectral data (boxes with dashed lines indicate inputs/outputs and boxes with solid lines indicate the analysis steps). Three different types of spectral data were used as inputs: (1) the original spectra, (2) spectra normalized to the peak intensity for each fluorescence spectrum or to the Soret valley (420 nm) for each diffuse reflectance spectrum, and (3) spectra normalized so that the area under the spectrum is unity.

Figure 2

Average peak-normalized fluorescence spectra for dysplastic tissues measured with the shielded and unshielded probe geometries at (a) the shortest excitation wavelength of 300 nm, and (b) one of the longer excitation wavelengths of 410 nm excitation.

Figure 2

Average peak-normalized fluorescence spectra for dysplastic tissues measured with the shielded and unshielded probe geometries at (a) the shortest excitation wavelength of 300 nm, and (b) one of the longer excitation wavelengths of 410 nm excitation.

Figure 3

The re-projected difference spectrum (dysplasia/CIS - normal) for the statistically most significant PC, PC3 derived from the peak-normalized spectra at 410 nm excitation and the actual difference spectrum (dysplasia/CIS - normal) of peak-normalized spectra at 410 nm excitation, both obtained with the unshielded probe geometry. The difference spectrum is calculated by subtracting point-by-point the average fluorescence intensity of normal tissues from the average fluorescence intensity of tissues diagnosed with dysplasia/CIS.

Figure 4

The detected fluorescence as a function of tissue depth for the shielded and unshielded probe geometries in a homogeneous tissue model with optical properties representative of epithelial and stromal layers at an excitation-emission wavelength pair of 410–460 nm (optical property set 1, Table 2).

Figure 5

The detected fluorescence as a function of tissue depth achieved with an illumination tilt angle of 0 and 45-degrees relative to the axis perpendicular to the tissue surface (for an illumination and collection fiber separation of 200 μm) in an epithelial tissue model (optical property set 2, Table 2). The dashed line at a depth of 450 μm separates the epithelium (E) and stroma (S) in the theoretical tissue model.

Figure 6

Sensitivity to the epithelial layer fluorescence as a function of illumination-collection fiber separation or center-to-center distance, at an angle of illumination of 0 (normal incidence), 15, 30 and 45 degrees.

Figure 7

Total fluorescence detected as a function of illumination-collection fiber separation or center-to-center distance, at an illumination angle of 0-degrees (for illumination and collection fiber NAs of 0.22) and at an illumination angle of 45-degrees (for illumination/collection fiber NA pairs of 0.22/0.22, and 0.22/0.37) (NA_i – numerical aperture of the illumination fiber, NA_c – numerical aperture of the collection fiber).